Sarang Burung Gua Niah Asli

www.LFI2U.blogspot.com

2009-03-30T11:47:00.001-07:00

Sarang Burung Gua Niah Asli

For English CLICK HERE

Untuk Bahasa Malaysia KLIK SINI

Laman Utama

2009-03-28T18:38:00.000-07:00

Maklumat Terkini LFI2U.blogspot.com

LFI2U mengadakan Promosi Sarang Burung Kolagen. Rebutlah peluang ini selagi stok masih ada.

Barisan orang kuat LenFa bergambar kenangan bersama pada hari pelancaran Program Usahawan Pemborong LenFa (19 April 2008)

About Lenfa

Tentang Lenfa

Kumpulan syarikat Lenfa ditubuhkan pada tahun 1967, bermula sebagai pengeluar barangan produk kesihatan herba tradisional China. Sehingga kini Lenfa menjadi peneraju dalam bidang pengeluaran dan kajian pembangunan (R&D) produk herba untuk pasaran global dan tempatan. Lenfa kini mengembangkan lagi sayap perniagaannya melalui penubuhan syarikat pengedaran produk kesihatan Lenfa iaitu Lenfa International (M) Sdn. Bhd. atau LFI.

Artikel : Lenfa Biotech sasar Pasaran Mesdaq akhir 2006

::sumber daripada Berita Harian, 2005-10-03

Program Usahawan Pemborong Lenfa

Menyedari produk berasaskan sarang burung semakin mendapat perhatian di kalangan masyarakat Bumiputera, LenFa International telah mengambil langkah proaktif dengan memperkenalkan konsep perniagaan yang mudah iaitu:

Program Usahawan Pemborong Lenfa

Program ini menonjolkan perniagaan yang lebih telus dan adil kepada para usahawan. Oleh itu LFI mengajak para usahawan yang berminat dalam perniagaan ini untuk sama-sama meraih pendapatan yang lumayan dengan konsep yang amat mudah.

TERIMA KASIH !

SIJIL HALAL JAKIM

SILA KLIK PADA IMEJ UNTUK LEBIH JELAS

Sijil Pengiktirafan & Akreditasi

SILA KLIK PADA IMEJ UNTUK LEBIH JELAS

About Lenfa

2009-03-28T18:37:00.000-07:00

The LenFa Group of Companies was established in 1967, growing from a ‘trading house’ to become a leading Manufacturer and Distributor of 'Health Care Supplements' and 'Traditional Herbal Chinese Medicine' in this region. Encouraged by our growing market and demands from overseas we have established 'Lenfa International' to meet our Mission in extending our products to 'Everyone, Everywhere'.

Lenfa International (M) Sdn. Bhd. or LFI will be expanding to countries around the Pacific Regions. We have carefully selected a range of Health Supplement Products which will bring health benefits to the consumers. Distributors will enjoy the benefit of the Marketing Plan which will bring about better Health & Financial Freedom to those who participate in the program.

With the support of our parent company, who are constantly in Research & Development of products to meet the demands of today’s health needs. Lenfa International will constantly upgrade and distribute products towards the well being of our consumers as well as a bigger market share for our distributors.

Mision & Vision

Optimism Live Life To The Fullest the company is committed to its vision in becoming a leading distributor of Health Supplements both in the local as well International markets, through its network of members worldwide. The company with the support of its parent company will constantly upgrade and manufacture health supplement products at competitive prices benefiting both consumers and its members.

The success of the company will be guided by its mission and total commitment to its members:

1.Provide a Rewarding and Achievable Business Opportunity.
2.Deliver Quality Products and Services.
3.Impart continuous Knowledge and instill Positive Values in our Network.
4.Provide a Conducive, Fun and Productive Workplace.
5.Maintain Integrity, Harmony and Honesty.

The Industry

Research & Development
Our relentless pursuit for continuous improvements led to the set up of a dedicated R&D team to observe and analyze the needs of the market, and subsequently apply the knowledge to develop better products and expand the range that Lenfa has to offer.

International Standards & Quality Assurance
We employ the latest technology in our state of the art manufacturing plant, which is accredited with ISO 9001:2000, ISO 22000:2005 and HACCP (Hazard Analysis Critical Control Point) certificates. Our stringent quality control from the selection of raw materials to production, storage and delivery has led us achieving the GMP (Good Manufacturing Practice) Standards. All our products are certified HALAL by Islamic Development Department of Malaysia (JAKIM).

HALAL CERTIFICATE (JAKIM)

CLICK ON THE IMAGE FOR CLOSE UP

Acreditations & Standardisations

CLICK ON THE IMAGE FOR CLOSE UP

Produk

2009-03-28T18:36:00.002-07:00

Imperial Concentrated Bird’s Nest

For thousands of years, bird's nest has been well known as an exotic royal's food in China. It has the ability to nourish the Yin, strengthen the kidney, alleviate the Yang, improve the spleen, moisturize and strengthen the lung, high contain of antioxidant to beautify as well as to prolong consumer's lifespan. Bird's nest is indeed a very valuable natural restorative food and appropriate for all age group.

The water-soluble protein in the bird’s nest which contain “Mitogenic Stimulation Factor” & “Epidermic Growth Factor” helps to promote cell regeneration, improves metabolism, regulates the endocrine system, therefore our skin will be improved naturally, it helps to moisturize & elastic skin and reduce wrinkles. Thus, it boosts up our body functions improves lung function, replenishes vitality, accelerates the recovery from diseases & strengthens the immun.

Advantages:
-Selected from high quality, 100% pure concentrated cave nest.
-Undergoes stringent procedure in eliminating the feathers (individually hand picked).
-Traditional stewing method plus advanced vacuum capping & sterilization method.
-Better texture & taste.
-Low sugar content..
-NO artificial preservative, colouring or flavour.

Ingredients: Bird’s Nest, Rock Sugar, RO water.

Contains: 150ml x 1 bottle.

Direction of Use: Consume one tablespoon, 2 times per day. Refrigerate after opening and finish within 14 days.

Penerangan Produk (BM)

Bird’s Nest + Collagen (BN 3+3)

(6bottles x 120ml)

(6bottles x 75ml)

Bird's Nest + Collagen ensures to moisturize, smooth & elastic skin, maintain eternal beauty, promote brain development, strengthen heart function, lower blood pressure, antioxidant, anti-aging, prevent cancer, aid for hair, joints, bone health, & prevent osteoporosis. It is used to strengthen the body, reinforce immunity, provide energy, enhance metabolism & suitable as a health supplements for everyone.

Advantages:
-100% pure Bloody Nest, Golden Silk Nest & Imperial White Nest.
-Fortified with collagens type I, II, & III from Deep Sea Fish Scales in Japan.
-Better texture & taste.
-Processed by using high pressure vacuum packed & short duration technique, no need to be refrigerated before open.
-Natural & pure ingredients.
-Selected from valuable & natural high quality of premium Sarawak Cave Nests..
-Confidence guarantee.
-
-NO artificial preservative, colouring or flavour.

Ingredients: Bird’s Nest, Collagens, Rock Sugar and Honey.

Contains:

Rainbow 3+3	:	6 bottles x 120ml per box
Bird's Nest 3+3	:	6 bottles x 75ml per box

Direction of Use: Recommended to consume one bottle per day with empty stomach in the early morning or before bedtime.

Penerangan Produk (BM)

Gamat Plus Cough Syrup

Sirap Ubat Batuk Gamat & Madu Plus

Indication:Traditionally used to relieve cough, to reduce phlegm, to relieve sore throat, hoarseness due to sore throat, body heatiness, fever and cold.

Advantages:
- Main function: FOR TREATMENT
- Helps to relief coughing & sore throat
- Helps to eliminate phlegm and soothing the throat
- Helps to cure for asthma and internal bleeding
- Helps to relief stress & fatigue
- Helps to reduce heatstroke

Ingredients: This product is produced from the finest selection of sea cucumber enriched with more than 10 types of traditional herbs plus pure honey. It is processed under strictly controlled and hygienic condition.

Contains: 150ml per bottle.

Direction of Use:

Adults : 1 tablespoon (10ml),
3 times per day

Children: 1 teaspoon (5ml),
3 times per day

Penerangan Produk (BM)

Ribose Chlorophyll

LFI Chlorophyll obtains its chlorophyll from fresh Alfalfa leaves. Alfalfa is known to be very rich in chlorophyll, at least 4 times higher than ordinary vegetables. No wonder when the Arabs of Persia first discovered the plant centuries ago, called it “Father of All Foods.”

Alpha Lipoic Acid (ALA) is naturally produced in the body whose production however diminishes as we age. ALA is an effective anti-oxidant that protects the body from the harmful effects of free radical. F ortified with selective D-R ibose will effective in promoting health.

Advantages:
-Amplifying the effects of other anti-oxidants including vitamin C and E.
-The increase of *Glutathione (GSH) level which helps with detoxification.
-The support of normal insulin level, glucose metabolism and sustain cellular energy metabolism.
-Strengthens memory and prevents brain aging.
-The decrease in chronic inflammation such as rheumatoid arthritis and inflammatory bowel disease.
-Power body cleanser.
-Inhibits bacterial growth.
-Improves oral health and hygiene.
-Raising the blood count in anemia.
-Detoxifying the liver.
-Relieves acne.
-Arthritic and rheumatic problems.
-For diabetic complications.
-Good for stomach ulcers.
-Good for the heart.
-Regulates the menstruation.
-Regulates the acid and alkaline.
-Improves blood circulation.
-Enhances kidney and bladder function.
-Improves appetite.
-Relieves insomnia.
-Helps constipation.
-Reduces fatigue.
-Alleviate muscles pain.
-Avoid cardiac muscle lack of blood and oxygen.
-Prevent heart disease.

Ingredients: 99.8% Liquid Chlorophyll Alfalfa, 0.1% Licorice, 0.1% D-ribose.

Contains: 500ml per bottle.

Direction of Use: Recommended to be consumed before meal three times per day with water, one tablespoon for children and two tablespoons for adult.

Penerangan Produk (BM)

i-Mann

It produced from the extracts of 18 types of selective herbs with high grade quality from China by using high technology. It promotes blood circulation, nourishes the kidneys and produces sperm, strengthens masculinity and arteries, increases blood supply.

Advantages:
-No hormone.
-No toxin and any side effect.
-Suitable for all kind of man and can be used as healthy food.
-Regulates body function and improves metabolism.
-Helps to improve heart, kidney and spleen function.
-Result effectiveness within 1 hour after consumption.
-Increase manliness energy.

Ingredients: Herba Epimedii, Herba Cistanches, Radix Angelicae, Cortex Eucommiae, Herba Cynomorii, Fructus Psoraleae, and etc.

Contains: 10 capsules per box.

Direction of Use: For adult 1 capsule per day (Recommended to consume after dinner, drink more water).

i-Nee

i-Nee is prepared by selected quality herbs with practicing traditional formulation and advance technology in extraction. Functional nutrients are retainable. Neither artificial stimulant, preservatives, flavouring nor colouring is added. This is the best ever found medicine for female, which is excellence remedy to improve quality of life.

It helps to smooth blood circulation, enhances body fitness for female, provides vitality, regulates menstrual health, detoxification, strengthens partly muscles, protects and shrinkage of uterus, maintains healthy of ovary, helps to reshape and revitalize the body, improves the firmness of skin tissues and ensures beautiful complexion. It also promotes metabolism and boosts up immune system, eliminates fatigue, regulates hormone, improves rheumatism, anemia and weakness of nervous system, balances blood pressure.

Advantages:
-Strengthens muscles of vagina.
-Anti-bacteria and anti-inflammatory.
-Strengthens kidney function and regulates menstrual health.
-Detoxification and maintain beauty.
-Maintains healthy of ovary.
-Protects and shrinkage of uterus.

Ingredients: Radix Angelicae, Rhimoza Ligustici, Radix Rehmanniae, Cortex Eucommiae, Ganoderma Lucidum, Pollen, Radix Paeoniae, and etc.

Contains: 6 bottles per box.

Direction of Use: Recommended to consume one bottle per day with warm water (Consume with meals, once per day for young ladies, twice per day for delivery women, drink more water after consumed).

100 i-MUM C%strum
(Extract Tablet / Extract Capsule / Extract Powder)

Colostrum-Nature’s First Food that last for life. Colostrum is the milky secretion produced by all mammals during the first after birth.

i-Mum Colostrum is collected only the first 4 milking upon the birth of the calf. It is the only true colostrum that offers maximum strength. It contains the bioactive compounds like lactoferrin, sphingolipids, probiotics, vitamins and minerals, also offering the highest source of immunoglobulin which is highly beneficial to our bodies. i-Mum Colostrum provides a perfect balance of both immune and growth factors, necessary to activate more than 50 processes in the body, ranging from immunity to regeneration and growth of all cell types. To cater for individual preference, i-Mum Colostrum is available in tablet, capsule and powder forms.

Advantages:
-Boosts up immune system.
-Detoxifies and purifies your body.
-Heals intestinal wall and mucosa.
-Regulates intestinal microflora.
-Fights viruses, bacteria, toxins, yeast and parasites.
-Increases your resistance to disease.
-Stimulated bone and muscle growth.
-Burns fat and rebuilds your body.
-Promotes fresher younger looking skin-relieves acne.
-Makes you look and feel younger.

Contains:

Tablet	:	60 tablets per bottle.
Powder	:	16 sachets per box.
Capsule	:	Contains 120 capsules per bottle.

Direction of Use:

Tablet	:	Take 2 chewable tablets twice per day.
Powder	:	Take one sachet (18g) daily.
Capsule	:	Take 2 capsules twice per day.

EZYLITE

This health care product is refined from Perillaseed. Function experiment proved that the product can regulate blood lipid. This product is suitable for people with Hyperlipaemaia.

Regulates blood lipids and blood pressure, slows down ageing, inhibits accumulation of platelets and prevents formation of blood clots.

Advantages:
-Major function: TREATMENT.
-Regulates blood lipid.
-Regulates blood pressure.
-Prevents formation of blood clots.
-Anti aging.
-Promotes blood circulation.

Ingredients: Perilla Oil, Edible Gelatin.

Contains: 90 capsules per bottle.

Direction of Use: Two times per day every morning and evening. Each times 3 capsules.

Piawaian Antarabangsa & Jaminan Kualiti
Bersesuaian dengan misi kami untuk mencapai inovasi untuk kehidupan yang lebih sihat & sejahtera, kami amat mementingkan aspek kualiti. Di LenFa kami menggunakan teknologi terkini di kilang kami yang diiktiraf ISO9001:2000, ISO22000 (2006) dan Hazard Analysis Critical Control Point (HACCP). Kawalan kualiti kami yang ketat bermula daripada proses pemilihan bahan mentah sehinggalah kepada pengeluaran, penyimpanan dan penghantaran. Ini telah membolehkan kami diiktiraf piawaian Good Manufacturing Practice (GMP). Kesemua produk kami telah disahkan HALAL oleh Jabatan Kemajuan Islam Malaysia (JAKIM) serta mendapat kelulusan daripada Kementerian Kesihatan Malaysia (MAL KKM).

SIJIL HALAL JAKIM

SILA KLIK PADA IMEJ UNTUK LEBIH JELAS

Sijil Pengiktirafan & Akreditasi

SILA KLIK PADA IMEJ UNTUK LEBIH JELAS

Beli Di Sini

2009-03-28T18:36:00.001-07:00

Dapatkan Sekarang!

Kaedah Dan Jumlah Pembayaran

Anda boleh membeli secara terus dengan stokis/pemborong kami yang berdekatan dengan anda.

Sila hubungi mereka secara terus dan dapatkan produk secara terus.

Business Manager :

M Haniff : 016-239 7779 (Hotline)
Bandar Mahkota Cheras

Stokis Negeri :

KEDAH : Puan Fariza Hassan : 017-472 5743

Pemborong:

Bangi (Bukit Mahkota): En Zainudin Mohd Jan - 012-911 4655
Kedah (Sungai Petani): Pn Fariza Hassan - 017-472 5743
Kuala Lumpur& Selangor: En M Haniff 016-239 7779 / 012-341 4112
Negri Sembilan: En Nor Hafizi Hamid 017-280 6670 / 016-367 1777
P. Pinang (Kepala Batas): Pn Hafizah Hassan - 014-259 6129

Atau Untuk pembayaran dan pembelian secara Online, sila ikuti langkah berikut:

Bayaran pembelian Produk boleh di buat sama ada menggunakan pemindahan wang secara online seperti maybank2u.com/CIMBclick ataupun mendepositkan tunai ke akaun melalui mesin Cash Deposit ATM mana-mana cawangan Maybank/CIMB di seluruh Malaysia.

Rujukan Harga :

Product Barangan	Contents Kandungan	Price Harga
BN 3+3 (75ml)	6 bottles / box	RM175
BN 3+3 (120ml)	6 bottles / box	RM252
Imperial Bird's Nest	150g / bottle	RM252
i-Mann	10 capsules / box	RM175
i-Nee	6 bottles / box	RM135
Tongkat Ali Ostrich Essence	6 bottles / box	RM99
i-Mum sachet	18g X 16 sachets	RM126
i-Mum tablet	60 tablets / bottle	RM126
Ribosa Chlorophyll	500ml / bottle	RM51
Natural Collagen Nutrients	20g X 20 sachets	RM123
Gamat Plus	1 X 150ml / bottle	RM19
Royal Jelly	1 X 200ml / bottle	RM26

Tunggu!!

Sila lihat PROMOSI kami untuk tawaran harga
yang lebih murah !!

Kos Penghantaran ditanggung sepenuhnya oleh pihak kami

Anda mesti bersetuju dengan terma dan syarat pembelian terlebih dahulu.

KLIK DI SINI untuk membaca terma dan syarat-syarat pembelian.

No. Akaun :

No akaun Maybank: 5620-8550-6549

ATAU

No akaun CIMB: 1210-0030729-52-5

Nama Pemegang Akaun:

Mohd Haniff bin Hassan

PERINGATAN

Sila pastikan No. Akaun dan Nama adalah betul sebelum anda meneruskan urusniaga. Pihak kami tidak akan bertanggungjawab atas sebarang kesilapan yang berlaku) Kemudian sila catatkan butiran maklumat berikut dan hantarkan kepada saya melalui SMS di talian :

016-239 7779

Nama Penuh
Alamat pos yang lengkap dan terkini (sebaik-baiknya bukan P.O Box).
E-mail yang sah (jika ada)
Masa, tarikh dan jumlah bank-in
Nama Produk
Kuantiti
Contoh : Haniff no 518a,kampung baru,08500 kota kuala muda,kedah,9.00pagi,2januari,rm38,gamat plusx2

dan sekiranya membuat online transfer, sila masukkan '3rd party e-mail address' dalam borang online maybank2u.com kepada :

aniptech@gmail.com

Kos Penghantaran ditanggung sepenuhnya oleh pihak kami

Penghantaran Produk Adalah Sulit Dan Dijamin Selamat

Adalah menjadi polisi kami untuk memastikan produk di hantar dalam keadaan selamat, sulit dan sampai ke tangan pembeli.

Bungkusan anda akan dihantar menggunakan servis berkualiti daripada Pos Laju dan di jamin sampai dalam masa selewat-lewatnya 5 hari.

Bertindak sekarang. Kesihatan, kecantikan, kekayaan dan kesejahteraan adalah hakmilik setiap insan.

TERIMA KASIH !

Hubungi Kami

2009-03-28T18:35:00.002-07:00

HUBUNGI KAMI

Anda boleh menghubungi stokis/pemborong berdekatan atau e-mel kepada kami dengan menggunakan borang di bawah atau kepada alamat seperti di bawah.

Haniff (Business Manager LFI)
Bandar Mahkota Cheras

HP :

016-239 7779 (Hotline)

E-mel ke :

aniptech@gmail.com

YM :

colsemetet@yahoo.com

Sekiranya anda gagal menghubungi saya melalui telefon, sila kirim pesanan anda melalui sms. Ini kerana saya mungkin sibuk. Saya akan menghubungi anda secepat mungkin.

Anda juga boleh menghubungi stokis/pemborong yang terdekat dengan anda untuk sebarang pertanyaan lanjut.

Senarai Stokis/Pemborong Lenfa International yang berdaftar dengan http://lfi2u.blogspot.com/

Stokis Negeri:

Kedah : Pn Fariza Hassan - 017-472 5743

Pemborong:

Bangi (Bukit Mahkota): En Zainudin Mohd Jan - 012-911 4655
Kedah (Sungai Petani): Pn Fariza Hassan - 017-472 5743
Kuala Lumpur& Selangor: En M Haniff 016-239 7779 / 012-341 4112
Negri Sembilan: En Nor Hafizi Hamid 017-280 6670 / 016-367 1777
P. Pinang (Kepala Batas): Pn Hafizah Hassan - 014-259 6129

Stokis & Pemborong Diperlukan!!!

LenFa International (LFI) mengajak para usahawan yang berminat dalam perniagaan ini untuk meraih pendapatan lumayan dalam konsep yang mudah. Menyedari produk berasaskan sarang burung semakin mendapat perhatian dari kalangan masyarakat Bumiputera, LenFa telah mengambil langkah proaktif dengan melancarkan:

"Program Usahawan Pemborong
Lenfa International (Usahawan LenFa) 2008"
(maklumat lanjut, KLIK SINI)

E-mail kami (Klik sini)

TERIMA KASIH !

Testimoni

2009-03-28T18:35:00.001-07:00

Testimonial 1
:: Mingguan Wanita 17 - 23 Oktober 2008

Klik pada gambar untuk imej lebih jelas

Testimonial 2
:: Forum mesra.net;

Saya buat kapsul sarang burung layang layang untuk makan sendiri. Sebab saya punya gedung sendiri sarang burung layang layang berwarna putih gred 1. Saya mengidap "heart blockage" asma, lelah, sengal sengal tulang, paru paru ber-air, sesak nafas, batuk kronik, kuat merokok.Doktor menasihatkan saya agar menjalani beberapa ujian menggunakan:- angiogram, city scan dan MRI scanning.

Sebelum menjalani ujian tersebut dalam masa 1 bulan saya amalkan memakan kapsul sarang burung layang layang. Saya proses sarang burung tersebut dengan membersihkan bulu bulu bercampur dalam sarang burung tersebut dan cuci bersih, selepas itu saya simpan dalam oven sekurang kurangnya 45 minit dengan tujuan untuk mematikan kuman kuman.

Setelah 1 bulan barulah saya menjalani ujian angiogram, city scan dan MRI scanning dihospital Kerajaan. Keputusan dari ujian tersebut saya tidak ada apa apa blockage disaluran jantung, paru paru saya tidak ada ber-air lagi, batuk kronik hilang, asma saya baik, tulang tulang saya tak ada ngilu-ngilu lagi dan muka saya licin tanpa ubat apa apa, ujian juga mendapati bahawa tidak terdapat tanda tanda saya seorang perokok, makan kapsul sarang burung telah menjadi amalan saya setiap hari.

Bila berita ini tersebar dari mulut kemulut, rumah saya jadi macam klinik dah....

Baca seterusnya di SINI...

Promosi

2009-03-28T18:34:00.002-07:00

Promosi #1

TAWARAN HEBAT

Dapatkan diskaun sehingga 50% apabila anda membuat pesanan melalui internet!!
Harga Promosi RM125 sekotak.

Sila klik sini untuk pesanan

PROMOSI BERGANDA

Dapatkan diskaun sehingga 50% apabila anda membeli 2 kotak Rainbow 3+3!!
Harga Promosi RM250 untuk 2 kotak.

Sila klik sini untuk pesanan

Promosi #2

TAWARAN TERHAD

Dapatkan diskaun sehingga 40% apabila anda membuat pesanan melalui internet!!
Harga Promosi RM100 sekotak.

Sila klik sini untuk pesanan

PROMOSI BERGANDA

Dapatkan diskaun sehingga 40% apabila anda membeli 2 kotak BN 3+3!!
Harga Promosi RM200 untuk 2 kotak.

Sila klik sini untuk pesanan

DAPATKAN PROMOSI INI SEKARANG JUGA SEMENTARA STOK MASIH ADA !

Hubungi Kami

2009-03-28T18:34:00.001-07:00

&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;ampamp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt;br&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;ampamp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;gt;

Soalan Lazim

2009-03-28T18:33:00.001-07:00

FAQs
Coming Soon!

Terma & Syarat

2009-03-28T18:32:00.000-07:00

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Polisi Hakmilik

2009-03-28T18:30:00.000-07:00

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Fakta Kandungan Kimia Dalam Sarang Burung

2009-03-27T20:30:00.001-07:00

Glycobiology Advance Access originally published online on October 11, 2006
Glycobiology 2007 17(2):157-164; doi:10.1093/glycob/cwl058

Occurrence of a nonsulfated chondroitin proteoglycan in the dried saliva of Collocalia* swiftlets (edible bird's-nest)*

Hiroki Nakagawa³^,4, Yoichiro Hama⁴, Toshihisa Sumi⁴, Su-Chen Li³, Karol Maskos³^,7, Kittiwan Kalayanamitra²^,5, Shuji Mizumoto⁵^,6, Kazuyuki Sugahara⁵^,6 and Yu-Teh Li¹^,3

³ Department of Biochemistry, Tulane University Health Sciences Center School of Medicine, New Orleans, LA 70112
⁴ Department of Applied Biological Sciences, Saga University, Saga 840-8502, Japan
⁵ Department of Biochemistry, Kobe Pharmaceutical University, Higashinada-ku, Kobe 658-8558, Japan
⁶ Laboratory of Proteoglycan Signaling and Therapeutics, Faculty of Advanced Life Science, Hokkaido University, Frontier Research Center for Post-Genomic Science and Technology, Kita 21-jo, Nishi 11-choume, Kita-ku, Sapporo 001-0021, Japan
¹ To whom correspondence should be addressed; Tel.: (504)-988-2451; Fax: (504)-988-2739; e-mail: yli1@tulane.edu

Received on June 19, 2006; revised on September 27, 2006; accepted on September 29, 2006

	Abstract

Despite their wide occurrence, proteoglycans (PGs) have neverbeen isolated from the saliva of higher animals. We found thatthe Collocalia glycoproteins isolated from edible birds'-nests(the dried forms of regurgitated saliva of male Collocalia swiftlets)were rich in a PG containing nonsulfated chondroitin glycosaminoglycans(GAGs). We have devised a method to isolate a PG from the waterextract of the white nest built by Aerodramus fuciphagus (whitenest swiftlets) with a yield of 2-mg PG per gram nest. ThisPG contained 83% of carbohydrates, of which 79% were GalNAcand GlcUA (D-glucuronic acid) in an equimolar ratio. By usingchondroitin AC lyase, the structure of GAGs in this PG was establishedto be chondroitin ( " border="0"> 4GlcUAß1 " border="0"> 3GalNAcß1" border="0"> )_n chains. The average molecular mass of the chondroitin chainwas estimated to be 49 kDa by gel filtration. We have isolateda linkage region hexasaccharide, HexUA1 " border="0"> 3GalNAcß1" border="0"> 4GlcUAß1 " border="0"> 3Galß1 " border="0"> 3Galß1 " border="0"> 4Xyl,from this PG by chondroitinase ABC digestion to show that theGAGs in this PG are also linked to the core protein throughthe common tetrasaccharide linker, GlcUAß1 " border="0"> 3Galß1" border="0"> 3Galß1 " border="0"> 4Xyl, found in various PGs. As water wasnot effective in extracting uronic acid-containing glycoconjugatesfrom the black nest built by black nest swiftlets (A. maximus),we used 4 M guanidium chloride and anion-exchange chromatographyin the presence of urea to extract and isolate about 30 mgof a chondroitin PG preparation from 10 g of the desialylatedblack nest. As the biological significance of chondroitin isstill not well understood, bird's nest should become a convenientsource for preparing this unique GAG to study its biologicalfunctions.

Key words: chondroitin / proteoglycan / glycosaminoglycan / bird's nest / saliva

	Introduction

Proteoglycans (PGs) are ubiquitous constituents of intracellular,pericellular, and extracellular matrices of higher animals.Each PG contains a core protein and covalently linked glycosaminoglycan(GAG) chains. The distribution, structure, and function of PGshave been the subject of intensive studies (Hassell et al. 1986;Kjellén and Lindahl 1991; Hardingham and Fosang 1992;Iozzo 1998; Prydz and Dalen 2000; Kolset et al. 2004). Exceptfor hyaluronic acid, all GAGs are sulfated and are linked toa core protein. The nonsulfated GAGs are not usually found inbiological materials, except in early stages of biosynthesisor when cultured cells are grown under the condition in whichthe sulfate donor is deprived (Humphries et al. 1989). It shouldbe noted, however, that nonsulfated chondroitin has been foundin the capsular K4 antigen of Escherichia coli O5:K4:H4 (Rodriguezet al. 1988) and in Caenorhabditis elegans (Yamada et al. 1999).Despite their wide distribution, PGs have never been isolatedfrom the saliva of higher animals.

Edible birds'-nests are the dried form of the nests made fromregurgitated saliva of male Collocalia swiftlets. They can bedivided into the white nest and the black nest. The white –nest,built by white nest swiftlets (Aerodramus fuciphagus), is composedof only the dried saliva. In contrast, the black nest, madeby black nest swiftlets (A. maximus), contains the birds' featherstogether with the saliva (Valli and Summers 1990). Collocaliaglycoproteins prepared from birds' nests are known for theirhigh sialic acid content (Howe et al. 1961). While studyingthe substrate specificity of the two sialidases isolated fromMacrobdella leeches (Li et al. 1990), we found that the crudeleech sialidase released a disaccharide from Collocalia glycoproteinsprepared from both the black and the white birds' nests. Bychemical analyses and NMR spectroscopy, the structure of thisdisaccharide released from the white nest Collocalia glycoproteinwas established to be _4,5HexUA1 " border="0"> 3GalNAc, which is identicalto that released from chondroitin by chondroitin AC lyase. Wereasoned that Collocalia glycoproteins of edible birds'-nestsmust contain a GAG composed of chondroitin, and that Macrobdellaleeches contained a lyase with the specificity similar to thatof microbial chondroitin AC lyase (Linhardt et al. 1986). Thispaper reports the isolation and characterization of a chondroitinPG from edible birds'-nests.

	Results and discussion

Characterization of an unknown compound released from white nest Collocalia glycoprotein by crude leech sialidase.

We found that in addition to Neu5Ac and 2,7-anhydro-Neu5Ac (Liet al. 1990), the crude sialidase prepared from the Macrobdellaleech released an unknown compound (UC) with thin layer chromatography(TLC)-mobility faster than that of Neu5Ac and 2,7-anhydro-Neu5Acfrom Collocalia glycoproteins prepared from either the blackor the white nest (Figure 1). We were intrigued by thefact that the crude leech sialidase released more UC than Neu5Acand 2,7-anhydro-Neu5Ac combined from the Collocalia glycoproteinprepared from the white nest as shown in Figure 1. We subsequentlycarried out the isolation of this UC and obtained 1.5 mgof pure UC from 250 mg of white nest Collocalia glycoproteinusing the procedure described under the section Methods. OnTLC plates, UC did not give a color when sprayed with resorcinol-HCl(Svennerholm 1957) or orcinol-H₂SO₄ (Winzler 1955), suggestingthat this compound was devoid of sialic acid and neutral sugar.In test tubes, UC reacted with the carbazole reagent (Galambos1967) to give a typical pink color for uronic acids. It alsogave a positive Elson–Morgan reaction (Gatt and Berman1966), suggesting the presence of a hexosamine. The ratio ofuronic acid to hexosamine in UC was found to be 1:1. On a TLCplate, UC reacted with iodine vapor to give a brownish color,indicating the possible presence of a double bond. This wasalso supported by the fact that UC had the UV absorption at230 nm. When analyzed by gas liquid chromatography (GLC),the hexosamine in the acid hydrolyzate of UC was identifiedto be galactosamine. The results of chemical analyses suggestthat UC contained an unsaturated uronic acid and GalNAc in 1:1ratio. The structure of UC was finally confirmed by NMR spectroscopy.As shown in supplementary material, the 500-MHz proton NMR chemicalshifts of the sugar residues in UC were found to be identicalto that of the authentic sodium salt of _4,5HexUA1 " border="0"> 3GalNAc (Yamadaet al. 1992).

Fig. 1. Liberation of an unknown compound (UC) from the Collocalia glycoprotein prepared from the white nest by a crude leech sialidase preparation. The reaction mixture contained 150 µg of Collocalia glycoprotein in 50 µL of 50 mM sodium acetate buffer, pH 5.5, and 0.2 units of crude leech sialidase (Li et al. 1990). After incubation at 37 °C for 17 h, 50 µL of ethanol was added to the incubation mixture to stop the reaction. The mixture was vortexed and centrifuged to remove the precipitated protein. The supernatant (30 µL) was evaporated to dryness and analyzed by TLC using n-butanol/acetic acid/water (2:1:1, v/v/v) as the developing solvent. The plate was sprayed with the diphenylamine–aniline–phosphoric acid reagent and heated at 110 °C for 15–30 min to reveal glycoconjugates (Anderson et al. 2000). C, the Collocalia glycoprotein prepared from the white nest; E, crude leech sialidase; UC, unknown compound; 2,7-AN, 2,7-anhydro-Neu5Ac.

Isolation and characterization of a chondroitin PG from the white nest

The liberation of _4,5HexUA1 " border="0"> 3GalNAc from the Collocalia glycoproteinprepared from the white nest by the crude leech sialidase indicatedthat this source contained a GAG with the structure consistentwith that of chondroitin. It also suggested that Macrobdellaleeches contained a chondroitinase AC-like lyase capable ofde-polymerizing the chondroitin chain. Since the uronic acid-containingglycoconjugates in the white nest were readily soluble in water,we devised a method to use Sepharose CL-6B filtration (Figure 2A),DEAE-Sephacel anion-exchange chromatography (Figure 2B),and Sephadex G-75 filtration (Figure 2C) to isolate approximately30 mg of chondroitin PG from the water extract preparedfrom 15 g of the white nest. As shown in Figure 2A,by Sepharose CL-6B gel filtration, sialic acid- and uronic acid-containingglycoconjugates were eluted before the main protein peak. SincePGs with uronic acid-containing GAGs are more negatively chargedthan sialoglycoconjugates, the anion-exchange chromatographyseparated the uronic acid-containing PGs from sialoglycoconjugates(Figure 2B). Sephadex G-75 filtration purified the majoruronic acid-containing glycoconjugate as shown in Figure 2C.Since chondroitin PG of white nest was highly soluble in water,we chose water as eluent to perform the gel filtration (Figure 2Aand C) for the facile isolation of this PG. Under this condition,chondroitin PG was retarded by the Sephadex G-75 column, dueto the interaction of the PG with the gel matrix as shown inFigure 2C. Thus, the elution position of the chondroitinPG shown in Figure 2C does not reflect its molecular size.Using this isolation scheme, we were able to isolate approximately30 mg of chondroitin PG from 15 g of white bird'snest. By Superdex-200 filtration, the average molecular massof the 2-aminobenzamide (2AB)-derivatized chondroitin GAG chainsderived from chondroitin PG was estimated to be 49 kDa,based on the elution position as calculated from a calibrationcurve obtained with authentic dextrans of various molecularsizes (Figure 3) . The constituent sugars in this PG preparationwere determined to be GalNAc (42%), GlcUA (37%), GlcNAc (1.4%),Gal (1.8%) and Xyl (0.6%). It is remarkable that this preparationcontained 83% of carbohydrates, of which 79% were GalNAc andGlcUA in equimolar ratio. This preparation was devoid of sialicacid and contained the following amino acids in mole%: Asp,11.8; Ser, 10.1; Thr, 7.1; Glu, 14.9; Gly, 8.8; Ala, 7.2; Val,9.0; Ile, 3.5; Leu, 8.7; Tyr, 3.1; Phe, 3.0; His, 3.1; Lys,4.2; and Arg, 5.6. Upon treatment of this preparation with 0.3 MNaOH at 25 °C for 63 h, 23% of Ser and 19% ofThr were destroyed, suggesting that these two amino acids arelinked to a sugar chain through the O-glycosidic linkage. Theinfrared spectrum of this preparation did not show the absorptionat 1230 cm^–1 (S = O), 850 cm^–1 found inC–O–S of chondroitin 4-sulfate and 820 cm^–1found in C-O-S of chondroitin 6-sulfate (Mathews 1958; Lloydet al. 1961). We were also not able to detect the presence ofsulfate in 0.5 mg of this PG preparation by using the rhodizonatemethod (Terho and Hartiala 1971). Chondroitin AC lyase liberated_4,5HexUA1 " border="0"> 3GalNAc from this preparation (Figure 4). Theseresults conclusively show that the GAGs in the PG prepared fromthe white nest are made up of chondroitin ( " border="0"> 4GlcUAß1" border="0"> 3GalNAcß1 " border="0"> )_n chains and that this PG is a chondroitinPG.

Fig. 2. Panel (A), Sepharose CL-6 B gel filtration of the aqueous extract of white nest. Panel (B), DEAE-Sephacel chromatography of the fractions containing uronic acid and sialic acid shown in panel A. Panel (C), Sephadex G-75 gel filtration of the uronic acid containing fractions shown in panel B. The horizontal bars indicate the fractions pooled. Uronic acid was determined by the carbazol reaction (Galambos 1967). Detailed experimental conditions are described under the section Methods.

Fig. 3. Determination of the molecular mass of the chondroitin GAG chains in bird's nest chondroitin PG by gel filtration on Superdex-200. The chondroitin GAG chains were released from bird's nest PG by treating the PG with 0.5 M LiOH overnight at 4 °C and labeled 2AB. The 2AB-derivatives of the GAG chains were analyzed by gel filtration using a Superdex-200 column (0.5 x 30 cm) with 0.2 M NH₄HCO₃ as the eluent. The arrows indicate the elution positions of authentic dextrans with different sizes. a, 65 000 Da; b, 37 500 Da; c, 18 100 Da; V₀, void volume; V_t, total column volume.

Fig. 4. Enzymatic liberation of _4,5HexUA1 " border="0"> 3GalNAc from bird's nest chondroitin PGs preparations. Each PG (150 µg) was incubated with 0.1 unit of chondroitin AC lyase in 50 µL of 0.01 M sodium phosphate buffer, pH 7.0 at 37 °C for 17 h. The reaction was stopped by the addition of 50 µL of ethanol to the reaction mixture. After centrifugation, 30 µL of the clear supernatant was evaporated to dryness and analyzed by TLC using the procedures described in Figure 1. W, PG from white nest; B, PG from black nest; E, chondroitin AC lyase; S, standard _4,5HexUA1 " border="0"> 3GalNAc (upper band) and _4,5HexUA1 " border="0"> 3GalNAc-4-sulfate (lower band).

Structural analysis of the linkage region hexasaccharide prepared from the white bird's nest chondroitin PG

It has been shown that exhaustive digestion of chondroitin andchondroitin sulfates with chondroitinase ABC (Yamagata et al.1968) yields 4,5-unsaturated disaccharide and the linkage region4,5-unsaturated hexasaccharide–peptide core (Sugaharaet al. 1988; Sakaguchi et al. 2001). We found that the rateof degradation of chondroitin by chondroitinase ABC was muchslower than that of chondroitin sulfates. Thus, as describedunder the section Methods, prolonged incubation with chondroitinaseABC was necessary for exhaustive digestion of bird's nest chondroitinPG to prepare the linkage region hexasaccharide. The putativelinkage region hexasaccharide prepared from the white nest chondroitinPG was set free from the core protein by LiOH (Sakaguchi etal. 2001), labeled 2AB, and analyzed by anion-exchange highperformance liquid chromatography (HPLC) on an amine-bound silicaPA-03 column according to Kinoshita and Sugahara (1999). The2AB-labeled chondroitin PG linkage region hexasaccharide waseluted at the position of the authentic unmodified linkage regionhexasaccharide (Figure 5B). A mixture of the authentic2AB-labeled linkage region hexasaccharides (Sakaguchi et al.2001) was mixed with the 2AB-labeled chondroitin PG linkageregion hexasaccharide fraction and examined by anion-exchangeHPLC (Figure 5C). The chondroitin PG linkage region hexasaccharidewas co-eluted with the unmodified authentic linkage region hexasaccharide.The 2AB-labeled chondroitin PG linkage region hexasaccharidewas digested with chondroitinase AC-II to yield a 2AB-labeledlinkage region tetrasaccharide. Upon anion-exchange HPLC analysis,the 2AB-labeled compound in the chondroitinase AC-II digestwas eluted in a position of the authentic unmodified linkageregion tetrasaccharide (HexUA1 " border="0"> 3Galß1 " border="0"> 3Galß1" border="0"> 4Xyl-2AB) (data not shown), confirming the structure of theparent linkage region hexasaccharide of the bird's nest chondroitinPG to be HexUA1 " border="0"> 3GalNAcß1 " border="0"> 4GlcUAß1 " border="0"> 3Galß1" border="0"> 3Galß1 " border="0"> 4Xyl. To further establish the structureof the isolated linkage region hexasaccharide, the delayed extraction(DE) matrix-assisted laser desorption ionization time-of-flightmass spectrometry (MALDI-TOF MS) was used to determine its molecularmass. About 30 pmol of the hexasaccharide was mixed with10 µg of the matrix, 2,5-dihydroxybenzoic acid, andanalyzed by MALDI-TOF MS in a positive ion mode using a VoyagerDE-RP/Pro (Perspective Biosystems, Framingham, MA) in a linearmode (Sakaguchi et al. 2001). The major molecular masses ofthe hexasaccharide were calculated to be 1033 and 1055, whichcorresponded to mono- and di-sodiated forms of the unmodifiedlinkage region hexasaccharide, respectively (Figure 6).By NMR analysis, the proton chemical shifts of the constituentsmonosaccharides of the linkage region hexasaccharide isolatedfrom bird's nest chondroitin PG were found to be identical tothat of the unmodified linkage region hexasaccharide isolatedfrom whale cartilage chondroitin sulfate PG (Figure 7).This analysis clearly established that the structure of thelinkage region oligosaccharide isolated from the bird's nestchondroitin PG is HexUA1 " border="0"> 3GalNAcß1 " border="0"> 4GlcUAß1" border="0"> 3Galß1 " border="0"> 3Galß1 " border="0"> 4Xyl.

Fig. 5. Anion-exchange HPLC of the linkage region hexasaccharide isolated from bird's nest chondroitin PG. The isolated putative linkage region hexasaccharide was labeled 2AB and analyzed by anion-exchange HPLC on an amine-bound silica column. Panel A, the 2AB-derivatives of the authentic linkage region hexasaccharides; Panel B, the putative linkage region hexasaccharide isolated from bird's nest chondroitin PG; Panel C, a mixture of the authentic linkage region hexasaccharides and the linkage region from bird's nest chondroitin PG. The elution positions of the authentic 2AB-labeled linkage region hexasaccharides are indicated in panel A by arrows. a, _4,5HexUA1" border="0">3GalNAcß1 " border="0"> 4GlcUAß1 " border="0"> 3Galß1 " border="0"> 3Galß1 " border="0"> 4Xylol; b, _4,5HexUA1 " border="0"> 3GalNAc(6S)ß1 " border="0"> 4GlcUAß1 " border="0"> 3Galß1 " border="0"> 3Galß1 " border="0"> 4Xylol; c, _4,5HexUA1 " border="0"> 3GalNAc(4S)ß1 " border="0"> 4GlcUAß 1 " border="0"> 3Galß1 " border="0"> 3Galß1 " border="0"> 4Xylol; d, _4,5HexUA1 " border="0"> 3GalNAc(4S)ß1 " border="0"> 4GlcUAß1 " border="0"> 3Gal(4S)ß1 " border="0"> 3Galß1 " border="0"> 4Xylol.(S) in these oligosaccharides means sulfate.

Fig. 6. DE MALDI-TOF mass spectrum of the linkage region hexasaccharide isolated from bird's nest chondroitin PG. Representative DE MALDI-TOF mass spectra of the bird's nest chondroitin PG linkage region were recorded in a positive ion mode with 2,5-dihydroxybenzoic acid as the matrix. Major molecular ion signals were assigned as indicated by arrows.

Fig. 7. Structural-reporter-group regions of one-dimensional 500-MHz ¹H NMR spectrum of the linkage region hexasaccharide isolated from the white bird's nest chondroitin PG. The numbers and letters refer to the corresponding residues in the structures. G, GalNAc or Gal; U, GlcUA; U, HexUA; X, Xyl. For anomeric protons, only numbers were used to designate each sugar residue.

Presence of chondroitin PG in the black nest

By TLC, the crude leech sialidase also liberated _4,5HexUA1 " border="0">3GalNAc from the collocalia glycoprotein prepared from the blacknest, suggesting the presence of a GAG with the structure consistentwith that of chondroitin in this nest. We found that water wasnot effective in extracting uronic acid-containing glycoconjugatesfrom the black nest. To prepare PGs from the black nest, wetreated the nest with 0.1 M H₂SO₄ at 100 °C for1 h to release sialic acids from sialoglycoconjugates followedby using 4 M guanidium chloride (Sajdera and Hascall 1969),and DEAE-cellulose anion-exchange chromatography in the presenceof 7 M urea in 0.05 M Tris buffer, pH 6.8 (Antonopouloset al. 1974), to extract and isolate PGs from the desialylatednest. By this procedure, the amounts of uronic acid-containingglycoconjugates eluted from the DEAE-cellulose column with 0.15 MNaCl and 2 M NaCl in 0.05 M Tris buffer, pH 6.8 containing7 M urea (Antonopoulos et al. 1974) were 142 mg and30.7 mg, respectively (Methods). The 0.15-M NaCl eluatewas found to contain 25% of carbohydrates. In addition to GlcUAand GalNH₂, this preparation also contained GlcNH₂, Gal, Man,Xyl, and iduronic acid, and the contents of Gal, GalNH₂, andGlcNH₂ were higher than that of GlcUA. In view of the heterogeneousnature of the sugar composition, this preparation must containseveral species of glycoconjugates. Thus, this preparation wasnot investigated further. The 2-M NaCl eluate, on the otherhand, contained 53% of carbohydrates. The major monosaccharidesfound in this preparation were GlcUA (20%) and GalNH₂ (23%),and the minor monosaccharides found were Gal (4.4%), Man (2.9%),and Xyl (1.3%). By infrared spectroscopy, this preparation wasalso found to be devoid of sulfate. As shown in Figure 4,chondroitin AC lyase also liberated _4,5HexUA1 " border="0"> 3GalNAc fromthe PG prepared from the black nest, indicating that this PGalso contained chondroitin chains. It should be pointed outthat the chondroitin PG from white nest was eluted from theDEAE-Sephacel column with approximately 0.2 M NaCl in water(Figure 2B), whereas the black nest chondroitin PG waseluted from the DEAE-cellulose column by 2 M NaCl in 0.05 MTris buffer, pH 6.8, containing 7 M urea as described underMethods. This could be due to the difference in the bindingavidity of DEAE-Sephacel and DEAE-cellulose under differentconditions. Antonopoulos, et al. (1974) used 2 M NaCl in0.05 M Tris buffer, pH 6.5–6.8 containing 7 Murea to elute PGs from the DEAE-cellulose column.

Through the observation of the enzymatic release of _4,5HexUA1" border="0"> 3GalNAc from the Collocalia glycoprotein, we have uncoveredthe presence of chondroitin PG in the dried regurgitated salivaof male swiftlets widely known as the edible bird's-nest. Wehave examined the Collocalia glycoproteins prepared from severalcommercial bird's nest preparations and found that all of themwere susceptible to chondroitin AC lyase to produce _4,5HexUA1" border="0"> 3GalNAc. The fact that we were able to isolate 30 mg ofa highly purified chondroitin PG preparation from 15 gof the white nest indicates that this PG is not a minor constituentof bird's saliva. The water extract prepared from 1 g ofwhite nest contained approximately 35 µmol of uronicacid and 11 µmol of sialic acid. Thus, the uronicacid content in the water extract of the white nest is threetimes higher than that of sialic acid, indicating that the bird'snest contains more chondroitin PG than sialoglycoconjugates.Most swiftlet nests are collected in caves on cliffs along theseacoast of South East Asia (Valli and Summers 1990). Afterswiftlets finish making their nests, it may take over a yearfor them to become commercially available. Thus, it is possiblethat the chemical compositions of the commercially availablebirds' nests have undergone partial degradation. There is nodoubt that the GAG chains and the core protein of chondroitinPG in these nests have also been partially degraded. It wouldbe interesting to isolate the intact chondroitin PG from thesalivary gland of live swiftlets.

Chondroitin was first isolated from bovine cornea by Davidsonand Meyer in 1954 (Davidson and Meyer 1954). Subsequently, thisGAG was also found in squid skin in 1964 (Anno et al. 1964)and in the capsular K4 antigen of E. coli O5:K4:H4 in 1988 (Rodriguezet al. 1988). Since then, chondroitin has not been found inother biological materials until the revelation of its presencein C. elegans in 1999 (Yamada et al. 1999). Chondroitin in C.elegans has been shown to be critically involved in cytokinesisof early embryogenesis (Mizuguchi et al. 2003). Although PGshave been isolated from various tissues, they have never beenisolated from the saliva of a higher animal. By analyzing theunsaturated disaccharides released by chondroitin AC lyase,rat and human saliva (Iversen et al. 1987; Okazaki et al. 1996)have been indirectly shown to contain chondroitin sulfate. Hyaluronicacid and chondroitin differs only in the hexosamine moiety ofthe disaccharide repeating units. Hyaluronic acid is usuallynot linked to a PG molecule. Our results indicate that the chondroitinchain and the core protein in the bird's nest chondroitin PGare also linked through the common tetrasaccharide linker, GlcUAß1" border="0"> 3Galß1 " border="0"> 3Galß1 " border="0"> 4Xyl, found in variousPGs (Rodén and Smith 1966; Hassell et al. 1986; Kjellénand Lindahl 1991; Hardingham and Fosang 1992; Iozzo 1998; Sugahara1998; Prydz and Dalen 2000; Ueno et al. 2001; Kolset et al.2004). It is intriguing that the saliva of swiftlets is so richin chondroitin-containing GAG. Although edible bird's-nest souphas been a culinary delicacy in China since ancient times, thebiological importance for the presence of such a high levelof chondroitin PG in swiftlet saliva remains to be elucidated.As the biological significance of chondroitin is still not wellunderstood, birds' nests should become a convenient source forpreparing this unique PG to study its biological functions.

	Materials and methods

Materials

The white nest was obtained from a Chinese food market in Yokohama,Japan, and the black nest was purchased from oriental food storesin Boston and San Francisco. Collocalia glycoproteins were preparedfrom both the white- and the black nests as described by Howeet al. (1961). Crude leech sialidase was prepared accordingto Li et al. (1990). The following were purchased from commercialsources indicated: _4,5HexUA1 " border="0"> 3GalNAc, DEAE cellulose (fastflow), chondroitin AC lyase, chondroitinase ABC (EC4.2.2.4,from Proteus vulgaris), neuraminidase from Clostridium perfringens(Pronase E, Sigma, St. Louis, MO); precoated silica gel-60 TLCplate (Merck, Darmstadt, Germany). Wakogel C-300 (silica gel;Wako Pure Chemical Industries, Ltd., Osaka, Japan); SepharoseCL-6B, DEAE-Sephacel, Sephadex G-75, Sephadex G-25 (superfine),Superdex-200 (Amersham Pharmacia Biotech Inc., Piscataway, NJ);amine-bound silica PA-03 column (YMC Co., Kyoto, Japan).

Methods
Isolation of a UC released from Collocalia glycoprotein

To isolate sufficient quantities of UC for structural characterization,250 mg of Collocalia glycoprotein prepared from the whitenest (Howe et al. 1961) was dissolved in 5.7 mL of 50 mMsodium acetate buffer, pH 5.5, and incubated with 0.3 mLof crude leech sialidase (Li et al. 1990) at 37 °Cfor 17 h. The reaction mixture was applied onto a SephadexG-25 column (2.5 x 100 cm) equilibrated with water. Thecolumn was eluted with water at 25 mL/h and 5-mL fractionswere collected. A 10-µL aliquot of each fraction was analyzedby TLC to detect mono- and oligo-saccharides, as described underthe section Analytical methods. This step separated UC togetherwith Neu5Ac and 2,7-anhydro-Neu5Ac from the Collocalia glycoproteinand other proteins. The fractions containing UC, Neu5Ac, and2,7-anhydro-Neu5Ac were pooled, lyophilized, redissolved in0.5 mL of n-butanol/acetic acid/water (2:1:1, v/v/v), andapplied onto a Wakogel C-300 silica gel column (1.3 x 20 cm)equilibrated with the same solvent. The column was eluted withthe same solvent at 10 mL/h, and 1.1-mL fractions werecollected. A 10-µL aliquot of each fraction was analyzedby TLC to locate the positions of UC, Neu5Ac, and 2,7-anhydro-Neu5Ac,as described. Under this condition, UC was eluted first andwell separated from Neu5Ac and 2,7-anhydro Neu5Ac. Those fractionscontaining UC were pooled and evaporated to dryness to obtainapproximately 1.5 mg of UC.

Isolation of a chondroitin PG from white nest

For the extraction of chondroitin PGs from the white nest, 15 gof the pulverized nest was soaked in 450 mL of water overnight,briefly homogenized with a Polytron homogenizer and centrifugedat 15 000 g for 30 min. The pellet was re-extractedwith 450 mL of water and the two extracts were combinedand lyophilized to obtain 1.4 g of white powder. This whitepowder was dissolved in 40 mL of water and 10 mL aliquotof this solution was applied onto a Sepharose CL-6B column (4.0x 71 cm) equilibrated with water. The column was elutedwith water at 35 mL/h and 7.9 mL-fractions were collected.As shown in Figure 2A, the fractions (fractions 39–56)eluted before the main protein peak were found to contain bothuronic acid and sialic acid. They were pooled and lyophilizedto obtain 240 mg of dried powder. This powder was dissolvedin 5 mL of water and applied onto a DEAE-Sephacel column(Cl^– -form, 2.8 x 36 cm) equilibrated with water.After washing with water, the column was eluted with a linearNaCl gradient from 0 to 0.3 M (Figure 2B). The uronicacid-containing fractions (fractions 116–126) (Figure 2B)were pooled, dialyzed against water to remove NaCl, and lyophilizedto obtain 75 mg of dried powder. This powder was dissolvedin 2 mL of water and applied onto a Sephadex G-75 column(1.8 x 74 cm) equilibrated with water. The column was elutedwith water at 25 mL/h and 2.8-mL fractions were collected.As shown in Figure 2C, the uronic acid-containing materialswere resolved by Sephadex G-75 filtration into one broad andone sharp uronic acid-containing peaks. The sharp peaks (fractions58–68) were pooled and lyophilized to obtain 7.6 mgof a chondroitin PG preparation.

Determination of the molecular mass of the chondroitin GAG chain in chondroitin PG

To estimate the molecular mass of the chondroitin GAG chain,the chondroitin-PG was treated with 0.5 M LiOH overnightat 4 °C to release the chondroitin chain, and was thenlabeled 2AB. The excess 2AB reagent was removed by paper chromatography(Kinoshita and Sugahara 1999). The molecular mass of the 2ABderivative of the free chondroitin chain was analyzed by gelfiltration on a Superdex-200 column (0.5 x 30 cm) with0.2 M ammonium hydrogen carbonate as the eluent at a flowrate of 0.3 mL/min.

Preparation of the linkage region hexasaccharide from white bird's nest chondroitin PG

To prepare the 4,5-unsaturated hexasaccharide–proteincore, 110 mg of white nest chondroitin PG was digestedunder an aseptic condition with 10 units (additional 4 unitswere added after 3 days) of chondroitinase ABC for 7 days at37 °C in 3 mL of 0.05 M Tris–HCl buffer,pH 8.0, containing 0.3 mg of bovine serum albumin. Afterheating the digest in a boiling water bath for 3 min, thedigest was applied onto a Sephadex G-25 column (1.8 x 100 cm)equilibrated with 0.15 M ammonium hydrogen carbonate. Thecolumn was eluted with the same solution at 6.5 mL/h and2.8 mL-fractions were collected. Fractions were monitoredby UV absorption at 232 nm and 280 nm for the 4,5-unsaturatedhexasaccharide–protein core and 10-µL aliquot ofeach fraction was also examined by TLC for the detection ofthe released _4,5HexUA1 " border="0"> 3GalNAc. The 4,5-unsaturated hexasaccharide–proteincore was eluted in fractions 36–48, whereas _4,5HexUA1" border="0"> 3GalNAc in fractions 54–60. Fractions 36–48 werepooled and lyophilized to obtain 6.9 mg of hexasaccharide–proteincore preparation. To release the putative linkage region hexasaccharide,this preparation was subsequently treated with 1 mL of0.5 M LiOH at 4 °C for 15 h (Sakaguchi etal. 2001). After neutralization with acetic acid, the reactionmixture was again subjected to Sephadex G-25 gel filtrationusing the conditions described. Fractions 54–59 were foundto contain an oligosaccharide by TLC. These fractions were pooledand lyophilized to obtain 2.6 mg of the putative linkageregion hexasaccharide.

Isolation of chondroitin PG from black nest

The pulverized black nest (10 g) was soaked in 200 mLof 0.1 M H₂SO₄ at 4 °C overnight, homogenizedwith a Polytron homogenizer, and heated at 100 °C for1 h to remove sialic acids from glycoconjugates. Aftercooling to room temperature, the mixture was adjusted to pH7.0 by dropwise addition of 2 M NaOH while stirring. Themixture was exhaustively dialyzed against water and lyophilizedto yield 9.5 g of dried asialo bird's nest powder. Thispowder was then extracted overnight with 280 mL of 4 Mguanidium chloride at 4 °C with gentle shaking (Sajderaand Hascall 1969). The mixture was centrifuged at 15,000g for30 min and the pellet was re-extracted as described. Thetwo extracts were combined, concentrated to 60 mL withan Amicon ultrafiltration cell using a PM 10 membrane, exhaustivelydialyzed against 7 M urea in 0.05 M Tris–HClbuffer, pH 6.8, and applied onto a DEAE-cellulose column (Cl^–form, 2.5 x 4.2 cm), which had been equilibrated with 7 Murea in the same buffer. After washing with the same buffercontaining 7 M urea to remove the unabsorbed materials,the column was eluted at 35 mL/h with 0.15 M NaClin the same buffer containing 7 M urea followed by 2 MNaCl in the same buffer containing 7 M urea (Antonopouloset al. 1974). Fractions of 7 mL were collected. The fractionseluted by 0.15 M NaCl and 2 M NaCl were pooled, dialyzedexhaustively against water, and lyophilized. The yields (driedweights) of 0.15-M NaCl and 2-M NaCl eluates were 142 mgand 30.7 mg, respectively. The 0.15-M NaCl eluate was foundto contain 25% of carbohydrates. In addition to GlcUA and GalNH₂,this preparation also contained GlcNH₂, Gal, Man, and Xyl, andalso iduronic acid (IdoUA), and the contents of Gal, GalNH₂,and GlcNH₂ were higher than that of GlcUA. In view of the heterogeneousnature of the sugar composition, this preparation must containseveral species of glycoconjugates. Thus, this preparation wasnot investigated further. The 2-M NaCl eluate, on the otherhand, contained 53% of carbohydrates with GalNAc and GlaUA inequimolar ratio.

Analytical methods

Amino sugars were determined by the Elson and Morgan reactionas modified by Gatt and Berman (1966). Uronic acids were determinedby the carbazole reaction as modified by Galambos (1967). Quantitativeanalysis of iduronic acid and glucuronic acid by GLC was performedaccording to the procedures of Inoue and Miyawaki (1975). Aminosugars and neutral sugars were also determined as their trimethylsilylderivatives by GLC after methanolysis and reacetylation, asdescribed by Kimura et al. (1994). Sialic acids were releasedfrom glycoconjugates by heating the samples with 0.1 MH₂SO₄ at 80 °C for 1 h and free sialic acids weredetermined by the modified periodate-thiobarbituric acid methoddescribed by Uchida et al. (1977). Monosaccharides and oligosaccharideswere also analyzed by TLC using silica gel-60 plates and n-butanol/aceticacid/water (2:1:1, v/v/v) as the developing solvent. The platewas sprayed with the diphenylamine–aniline phosphoricacid reagent and heated at 110 °C for 15–20 min(Anderson et al. 2000). Amino acid composition of protein sampleswere determined by using a JOEL automatic amino acid analyzerafter hydrolyzing the protein samples with 6 M HCl at 110 °Cfor 24, 48, and 72 h under vacuum. For NMR spectroscopy,the sample (1.5 mg) was repeatedly exchanged with D₂O withintermittent lyophilization and dissolved in 0.5 mL ofD₂O. The ¹H NMR spectra of the UC isolated from Collocalia glycoproteinwere recorded at 25 °C on a GE Omega PSG 500 NMR spectrometer.The 500-MHz ¹H NMR spectra of the hexasaccharide prepared fromthe bird's nest chondroitin PG was recorded in a Varian VXR-500spectrometer at a probe temperature of 26 °C. Chemicalshifts were measured relative to acetone (2.225) in D₂O. Infraredabsorption spectra were recorded using a JOEL FT-IR on potassiumbromide disc containing 0.2% of the sample.

	Supplementary data

The supplementary figure shows the 500-MHz ¹H NMR spectra ofthe authentic sodium salt of _4,5HexUA1 " border="0"> 3GalNAc and the unknowncompound. Supplementary data are available at Glycobiology online(http://glycob.oxfordjournals.org).

	Acknowledgments

This work was supported by National Institutes of Health GrantNS 09626 (to Y.-T.L.) and by a Grant-in-Aid for Scientific Research-B16390026, the Human Frontier Science Program, and the Core Researchfor Evolutional Science and Technology (CREST) Program of theJapan Science and Technology (JST) Agency (to K.S.).

	Footnotes

² Present address: Department of Biochemistry, Faculty of Medicine,Chiang Mai University, Chiang Mai 50200, Thailand

⁷ Deceased, March 29, 2004.

None declared.

	Abbreviations

2AB, 2-aminobenzamide; DE, delayed extraction; GAG, glycosaminoglycan; GlcUA, D-glucuronic acid; GLC, gas liquid chromatography; MALDI-TOF MS, matrix-assisted laser desorption ionization time-of-flight mass spectrometry; HPLC, high performance liquid chromatography; PG, proteoglycan; TLC, thin-layer chromatography; UC, unknown compound.

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