Genipin

 

 

 

 

 

Abstract

 

Genipin is a hydrolytic product of geniposide, which is found in the fruit of Gardenia jasminoides Ellis.  The components of the fruit have been used in traditional Chinese medicine and as a blue colorant by food industries in East Asia.  The structure of genipin was discovered in the 1960’s.  Because it is a naturally occurring, biodegradable molecule with low cytotoxicity, genipin has recently been investigated as a crosslinking material in many biological applications.  Recent explorations into the use of genipin crosslinked gelatin for the use as a bioadhesive, wound dressing, and as bone substitutes, have shown it to have potential as a new and safe crosslinking agent.  The most common crosslinking agent has been glutaraldehyde, but because of concerns about its toxicity new methods are being tested like genipin.  In the area of forensic science, genipin is being examined as a new way of developing latent fingerprints on paper products.  Because it is an environmentally friendly natural product it shows great potential over the current reagent being used, ninhydrin. 

 

 

General Information

 

Genipin is an aglycone derived from geniposide, which is found in the fruit of Gardenia jasminoides Ellis.  Djerassi and his colleagues discovered the structure of Genipin in the 1960’s using NMR spectroscopic data and chemical degradation experiments.  It possesses the molecular formula C11H14O5 and contains a dihydropyran ring.1  Genipin itself is colorless but it reacts spontaneously with amino acids to form blue pigments.2  The blue pigments are edible and are currently being used as a blue food colorant in East Asia.3  The structure of Genipin is shown in Figure 1 along with other characteristics. 

 

 

Text Box: Figure 1: Structure of Genipin4 Molecular Formula: C11H14O51

Molecular Weight: 226.227 4

Melting Point: 120-121 °C 2

Systematic Name: Cyclopenta(c) pyran-4-carboxylic acid, 1,4a-alpha,5,7a-alpha-tetrahydro-1-hydroxy-7-(hydroxymethyl)-, methyl ester4

Toxicity: Mouse LD50 intravenous 153mg/kg and Mouse LD50 oral 237mg/kg4

Registry Number: 6902-77-84

 

 

 

 

 

 

 

 

 

Text Box: Figure 2: Gardenia jasminoides Ellis6 Natural Source

 

The fruit of Gardenia jasminoides Ellis (Figure 2) is the natural plant source of geniposide.  It is from the plant family Rubiaceace and is native to China.  It is an evergreen shrub that grows four to eight feet tall and wide.  The fruit are oval in shape about one-half inch to one inch long and orange in color.  The fruit of Gardenia jasminoides Ellis has been used as a tradition Chinese medicine to treat irritability in febrile diseases, jaundice, acute conjunctivitis, epistaxis, hematemesis, pyrogenic infections and ulcers of the skin, and externally on sprains and painful swelling due to blood stasis.5  The major component of the fruit of Gardenia jasminoides Ellis is the iridoid glycoside geniposide (Figure 3).3 

 

Text Box: Figure 3: Structure of Geniposide4

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Isolation of Geniposide and Genipin

 

Endo and Taguchi isolated geniposide and genipin using the following method in 1973.  This method is still used however, some modifications have been made.  Figure 5 shows the reaction.

 

1.     The Gardenia jasminoides Ellis fruits are crushed.

2.     The crushed fruit is then extracted with CHCl3 to remove the fats and oils and then extracted with MeOH, three times at room temperature.

3.     The combined MeOH extracts are then concentrated under reduced pressure until it is dry.  The result of these steps is a brown colored mass.

4.     The MeOH extracts are then chromatographed on charcoal using water and 10% aqueous EtOH to separate the sugars.  Then it is chromatographed using MeOH to separate the glycosides. 

5.     The glycosidic fraction eluted with MeOH is then rechromatographed on silica gel using a mixture of MeOH and CHCl3.  Geniposide is eluted with a mixture of 7 to 10% MeOH- CHCl3.  The residue is crystallized from acetone and forms colorless crystals.

6.     Geniposide and a solution of 1% aqueous solution of β-glucosidase are added to an acetate buffer (pH 5.0).

7.     This mixture is allowed to stand at 37°C for three hours.

8.     The reaction mixture is extracted with ether.

9.     The extract is concentrated and crystallized with ether-MeOH to yield colorless genipin.7

 

                                            

            Geniposide                                                                   Genipin

 

Figure 4:  Hydrolysis of Geniposide to produce Genipin.  Glucose is removed from geniposide.

 

 

 


β-glucosidase

 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

Genipin and Biomaterials

 

          Gelatin based materials have recently been studied as a biomaterial for use in bioadhesives, wound dressing material, and bone substitute.  Gelatin is a natural polymer which has a low antigenicity and is biodegradable.  However, gelatin does possess one important limitation.  It rapidly dissolves in aqueous environments, this leads to quick degradation at body temperature.  One method to improve gelatin is by treating it with crosslinking materials.  Chemical crosslinking is normally achieved by creating bonds between functional groups of amino acids.  The most popular chemical crosslinking reagent is glutaraldehyde, a synthetic reagent.8  Recently, reports of the cytotoxicity of glutaraldehyde due to the release of formaldehyde upon degradation has prompted research into the development of a natural crosslinking reagent.  For this reason, genipin which has low cytotoxicity and is naturally occurring has been investigated as a new crosslinking reagent for gelatin.9 

 

Genipin’s Crosslinking Mechanism

 

The crosslinking mechanism of genipin with gelatin and molecules containing primary amines is not well understood at this time and is still under investigation.  Touyama’s group proposed a mechanism for the reaction of genipin with a methylamine.  His group projected that the reaction occurred through a nucleophilic attack of the primary amine on the C3 carbon of genipin.  This caused an opening of the dihydropyran ring.  An attack on the resulting aldehyde group by the secondary amine group then followed.8  This reaction is shown below.10

Text Box: Figure 5: Touyama’s groups presumed mechanism for the reaction of Genipin with Methylamine10

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

The proposed reaction mechanism with genipin and methylamine is also believed to be the reaction mechanism for the reaction of genipin with molecules that contain a primary amine group such as gelatin.  The final step in the formation of the crosslinking material is believed to be dimerization produced by radical reactions.  This indicates that genipin can be used to form intramolecular and intermolecular crosslinks that have a heterocyclic structure with materials that contain a primary amine group.11 

 

Bioadhesives and Wound-Dressing Materials

 

Bioadhesives are used in surgery for tissue adhesion and also for stopping the flow of blood.  They are important for sealing air leaks and body fluid links in organs and tissues that are resistant to suture or stapling techniques.  The ideal bioadhesive according to Otani and his colleagues should have reasonable bonding strength to the tissue, it should be viscous enough that it can be applied to the tissue area without following to other areas of the tissue, the bioadhesive must also be flexible enough to adhere to the tissue, and finally the bioadhesive must be biodegradable and nontoxic.12  The current bioadhesive being used is gelatin crosslinked with glutaraldehyde.12  However, as stated earlier the toxicity of glutaraldehyde has led to the investigation of the use of other crosslinking reagents.  Sung and his co-workers conducted an experiment to evaluate gelatin crosslinked with genipin glue to create a bioadhesive.12  They used the gelatin crosslinked with glutaraldehyde glue has a control for comparison.  They compared the cytotoxicity, the gelation time, bonding strength, and flexibility.  They found gelatin crosslinked with genipin glue to be much less cytotoxic about 10,000 times less then gelatin crosslinked with glutaraldehyde glue.  The gelation time for the gelatin crosslinked with genipin glue was longer than that of the gelatin crosslinked with glutaraldehyde glue.  The bonding strength of the gelatin crosslinked with glutaraldehyde glue was greater then the gelatin crosslinked with genipin glue but still comparable.  The gelatin crosslinked with genipin glue was found to be more flexible than the gelatin crosslinked with glutaraldehyde glue. 

Sung and his co-workers also conducted an in vivo experiment to test the ability of gelatin crosslinked with genipin glue as a bioadhesive for closing skin wounds.9 The healing of skin wounds is currently done by the use of sutures but sutures increase the risk of infection and require the use of local or general anesthesia.  The in vivo test compared the use of sutures, gelatin crosslinked with glutaraldehyde glue, and gelatin crosslinked with genipin glue.  They found that the tensile strength of the skin across the wounds increased with time for both the glues and the suture.  All three techniques did not interfere with the healing of the wound and no calcification was seen.  It took six days for the suture to be reabsorbed while it took nine days for the gelatin crosslinked with genipin glue to be reabsorbed.  The gelatin crosslinked with glutaraldehyde glue took fourteen days to be completely reabsorbed.  The healing process of the suture was more rapid then the glues.  The wounds treated with gelatin crosslinked with genipin glue healed faster and less inflammation was seen then in the wound treated with gelatin crosslinked with glutaraldehyde glue.  Both of these experiments, indicate that further investigation into the use of gelatin crosslinked with genipin glue as a bioadhesive is warranted.

The results of the previous two studies encouraged Chang and his colleagues to develop a genipin crosslinked gelatin membrane to use as a wound dressing material.11  They used a glutaraldehyde crosslinked gelatin membrane as the control.  Their in vitro study indicated that the degree of crosslinking for both was equivalent.  The tensile strength of the genipin crosslinked gelatin membrane was much greater than the glutaraldehyde crosslinked gelatin membrane; however, the strain at fracture was much less in the genipin crosslinked gelatin membrane than the glutaraldehyde crosslinked membrane.  The swelling ratio of the genipin crosslinked membrane was smaller then the swelling ratio for the glutaraldehyde crosslinked gelatin membrane.  The genipin crosslinked membrane degraded at a slower rate then the glutaraldehyde crosslinked gelatin membrane when exposed to collagenase solution.  This can be attributed to the higher stereohindrance of the cyclic genipin crosslinked gelatin membrane.  In the in vivo study, they found that the inflammation in the genipin crosslinked gelatin membrane was less severe throughout the course of the experiment which was 21 days.  This indicates the genipin crosslinked gelatin membrane has a better biocompatibility then the genipin crosslinked gelatin membrane.  The healing rate of the wound using the genipin crosslinked gelatin membrane was also much faster then the healing time of the wound for the genipin crosslinked gelatin membrane.

 

Bone Substitute

 

The ability to repair bone defects still presents many challenges.  Bone defects can be various sizes and shapes caused by trauma, tumors, or infections.  In bone transplantation, bone from somewhere else in the body can be used but this has the disadvantage of limited bone supply and donor site morbidity.  Bone can also be transplanted form another individual but this can cause an immune response and can transmit diseases.  Therefore, synthetic bone-promoting materials are needed to repair bone defects.  The ideal bone composite material would possess properties such as good biocompatibility, good biodegradation, and be able to get cells into the defect site and promote new bone formation.13  Tricalcium phosphate has been investigated as bone substitute.  However, because it is granular it is hard to shape to the defect site on the bone.  Therefore, studies and investigations have been conducted using material composed of genipin crosslinked gelatin and tricalcium phosphate.13  Yao and his co-workers evaluated the use of a genipin crosslinked gelatin mixed with tricalcium phosphate bone substitute in an in vivo study using rabbit calvarial bone.13  In this study they found that the mixture was biocompatible; there was no inflammation found in the tissue below the mixture.  The mixture was also malleable and was easy to mold to the shape and size of the defect.  It also was biodegradable and aided in the growth of new bone through the release of gelatin and Ca2+.  The study also indicated that growth of new bone was occurring in the defected area and that the mixture of tricalcium phosphate and genipin crosslinked gelatin was being replaced by the new formed bone.  Their study indicates the genipin crosslinked gelatin mixed with tricalcium phosphate can be valuable as a bone substitute when repairing bone defects.

 

Genipin as a Fingerprint reagent

 

          Genipin is also being investigated in the field of forensic science as a fingerprint reagent to develop latent fingerprints on paper products.  In an article by Lee and his collogues, they investigated the use of genipin as a way to development amino acid stains.14  They compared genipin’s ability to develop amino acid stains with ninhydrin’s ability.  The product of the reaction of genipin with amino acids was very stable and lasted 7 months, but the ninhydrin product disappeared much sooner than the genipin product.  The sensitivity was also greater with genipin then ninhydrin as seen through the molar absorptivities.  Because of the properties genipin showed in this study, Almog and collogues investigated genipin as a fingerprint reagent.15  Ninhydrin is the current reagent used to develop latent fingerprints on paper products.  Almog and co-workers define an ideal reagent for developing latent fingerprints as a reagent that produces color and also fluorescent.  It must also be a simple reaction that can be done easily at a crime scene.  Lastly, it must be highly sensitive.  They found that the fingerprints developed with genipin were blue in color and showed clear ridge detail.  They were also able to visualize weak fingerprints using fluorescence and genipin shifted the excitation-emission domain toward the longer wavelength which produces less background.  They concluded that genipin meets the requirements for a fingerprint reagent and should be considered as an alternate to ninhydrin.  In a second study, Almog and co-workers showed the advantages of genipin over ninhydrin.16  Genipin has the advantage of the combination of both color and fluorescence in a single reaction and as stated early it has a low background when fluoresced because it is viewed at longer wavelengths.  Genipin is also safe and environment friendly because it is a natural plant product.  The safety of genipin is very important because ninhydrin has recently been reported to potentially cause health hazards.  Besides the cost of genipin they concluded that it has great potential to be used as a fingerprint reagent. 

 

Conclusion

 

Genipin is a naturally occurring material that shows great potential as a use in biomaterials and also as a fingerprint reagent.  As stated earlier genipin crosslinked material has been use as a bioadhesive, in wound dressing materials, and as a bone substitute.  In addition to these studies, studies have also been conducted to investigate genipin crosslinked gelatin as a conduit for peripheral nerve regeneration17 and genipin crosslinked chitosan for protein drug delivery18.  Based on these novel experiments, I believe that genipin will become an important crosslinking reagent for biomaterials in the future.  I also believe it can be valuable as a reagent to develop latent fingerprints.

 

References

1. Djerassi C., Nakano T., James A.N., Zalkow L.H., Eisenbraun E.J., Shoolery J.N. Terpenoids. XLVII. The Structure of Genipin. J. Org. Chem. 1961, 26, 1192-1206.

2. Djerassi C., Gray J.D., Kincl F.A. Naturally Occurring Oxygen Heterocyclics. IX. Isolation and Characterization of Genipin. J. Org. Chem. 1960, 25, 2174-2177.

3. Park J.E., Lee J.Y., Kim H.G., Hahn T.R., Paik Y.S. Isolation and Characterization of Water-Soluble Intermediates of Blue Pigments Transformed from Geniposide of Gardenia Jasminoides. J. Agric. Food Chem. 2002, 50, 6511-6514.

4. Natural Library of Medicine. Specialized Information Services. ChemIDplus Advanced. Full Record Genipin. http://chem.sis.nlm.nih.gov/chemidplus/jsp/common/ChemFull.jsp?MW=226.227 (accessed 03/02/2006).

5. Tsai T.R., Tseng T.Y., Chen C.F., Tsai T.H. Identification and Determination of Geniposide Contained in Gardenia Jasminoides and in Two Preparations of Mixed Traditional Chinese Medicines. J. Chromatogr. A. 2002, 961, 83-88.

6. Ishikawa S. Picture Book of Jia-Wei-Gui-Pi-Tang. http://web1.incl.ne.jp/ishikawa/PET/pict.html (accessed 03/09/2006).

7. Endo T., Taguchi H. The Constituents of Gardenia Jasminoides Geniposide and Genipin-getiobioside. Chem. Pharm. Bull. 1973, 21, 2684-2688.

8. Yao C.H., Liu B.S., Chang C.J., Hsu S.H., Chen Y.S. Preparation of Networks of Gelatin and Genipin as Degradable Biomaterials. Materials Chemistry and Physics 2004, 83, 204-208.

9. Sung H.W., Huang D.M., Chang W.H, Huang L.L.H., Tsai C.C., Liang I.L. Gelatin-derived Bioadhesives for Closing Skin Wounds: An in vivo Study. J. Biomater. Sci. Polymer Edn. 1999, 10, 751-771.

10. Touyama R., Inoue K., Takeda Y., Yatsuzuka M., Ikumoto T., Moritome N., Shingu T., Yokoi T., Intuye H. Studies on the Blue Pigments Produced from Genipin and Methylamine. II. ON the Formation Mechanisms of Brownish-Red Intermediates Leading to the Blue Pigment Formation.  Chem. Pharm. Bull. 1994, 42, 1571-1578.

11. Chang W.H., Chang Y., Lai P.H., Sung H.W. A Genipin-Crosslinked Gelatin Membrane as Wound-Dressing Material: in vitro and in vivo Studies. J. Biomater. Sci. Polymer Edn. 2003, 14, 481-495.

12. Sung H.W., Huang D.M., Chang W.H., Huang R.N., Hsu J.C. Evaluation of Gelatin Hydrogel Crosslinked with Various Crosslinking Agents as Bioadhesives: In vitro Study.  J. Biomed. Mater. Res. 1999, 46, 520.

13.Yao C.H., Lui B.S., Hsu S.H., Chen Y.S. Cavarial Bone Response to Tricalcium Phosphate-Genipin Crosslinked Gelatin Composite. Biomaterials 2005, 26, 3065-3074. 

14. Lee S.W., Lim J.M., Bhoo S.H., Paik Y.S., Hahn T.R. Colorimetric Determination of Amino Acids Using Genipin from Gardenia jasminoides. Analytica Chimica Acta 2003, 480, 267-274. 

15. Almog J., Cohen Y., Azoury M., Hahn T.R. Genipin-A Novel Fingerprint Reagent with Colorimetric and Fluorogenic Activity. J. Forensic Sci. 2004, 49, 255-257.

16.  Levinton-Shamuilov G., Cohen Y., Azoury M., Chaikovsky A., Almog J Genipin, a Novel Fingerprint reagent with Colorimetric and Fluorogenic Activity, Part II: Optimization, Scope, and Limitations. J. Forensic Sci. 2005, 50, 1367-1371.

17. Chen, Chang, Cheng, Tsai, Yoa, Lui An in vivo Evaluation of a Biodegradable Genipin-Cross-Linked Gelatin Peripheral Nerve Guide Conduit Material.  Biomaterials 2005, 26, 3911-3918.

18. Chen, Wu, Mi, Lin, Yu, Sung A Novel pH-Sensitive Hydrogel Composed of N,O-carboxymethyl Chitosan and Alginate Cross-linked by Genipin for Protein Drug Delivery. J. Controlled Release 2004, 96, 285-300.

 

BY: BRENDA VAANDERING