Microbial transformation of geniposide in Gardenia jasminoides Ellis into genipin by Penicillium nigricans

Available online at www.sciencedirect.com

Enzyme and Microbial Technology 42 (2008) 440–444

Microbial transformation of geniposide in Gardenia jasminoides Ellis into genipin by Penicillium nigricans Mengmeng Xu a , Qun Sun a , Jian Su a , Jianfang Wang a , Chun Xu a , Tao Zhang b , Qiling Sun a,∗ a

College of Life Sciences, Key Laboratory of Bio-resources and Eco-environment of Ministry of Education, Sichuan University, 29# Wangjianglu Street, Chengdu, Sichuan 610064, PR China b College of Life Science & Biotechnology, Shanghai Jiao Tong University F0408004, Shanghai 200240, PR China Received 8 November 2007; received in revised form 4 January 2008; accepted 7 January 2008

Abstract A filamentous fungi strain, Penicillium nigricans, producing ␤-glucosidase was screened to transform geniposide in Chinese traditional medicine Gardenia jasminoides into genipin, a highly efficient anti-inflammatory, anti-angiogenesis compound used in treatment of liver fibrosis also. By modulating the fermentation process, including fermentation temperature, gardenia concentration, rotation speed and medium capacity, along with monitoring mycelial biomass, residual sugar concentration (RSC), ammoniacal nitrogen concentration (ANC), amount of substrate geniposide, and ␤-glycosidase activity the conversion rate of geniposide into genipin could reach 95%. Under the all-over feedback control system, genipin was isolated by macroporous resin and then purified by silica gel column with the final purity over 98%. Purified product was identified as genipin by high performance liquid chromatography (HPLC), ultraviolet (UV), nuclear magnetic resonance spectroscopy (NMR), infrared absorption spectrometry (IR) and mass spectrometry (MS). © 2008 Elsevier Inc. All rights reserved. Keywords: Gardenia jasminoides; Penicillium nigricans; Geniposide; Genipin; Microbial transformation

1. Introduction The fruit of gardenia (Gardenia jasminoides Ellis), a traditional Chinese medicine, has long been used for the treatment of inflammation, jaundice, and hepatic disorders. Geniposide is one of the major iridoid glycoside compounds existing in the fruit of gardenia, being responsible for gardenia’s pharmacological activities of hepatic protection and anti-inflammatory effect. Mie et al. [1] used gardenia for the treatment of liver cirrhosis and their investigation showed that the activated hepatic stellate cells could be suppressed by genipin [1]. The pharmacokinetics studies suggested that geniposide, when it is orally administered, hydrolyzed into genipin by enzymes produced by intestinal bacteria [2]. It is genipin, but not geniposide, that functions as the main bioactive compound to exhibit the pharmacological activities of gardenia [3]. Furthermore, genipin is more

∗

Corresponding author. Tel.: +86 28 8541 0165; fax: +86 28 8541 5300. E-mail address: [email protected] (Q. Sun).

0141-0229/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.enzmictec.2008.01.003

effective against mutagenesis than other iridoid compounds. It has been proved that genipin not only reveals remarkable effects of anti-inflammatory and anti-angiogenesis, but also possesses the abilities of inhibiting lipid peroxidation and the production of nitrogen monoxide (NO), as well as protects hippocampal neurons from the toxicity of Alzheimer’s amyloid ␤ protein [4]. Additionally, genipin is a natural cross-linking agent with advantages of low toxicity and high biocompatibility. Thus it has been widely applied in a variety of medical fields such as nerve regeneration [5] and drug delivery [6]. Accordingly, genipin is a more favorable glucoside compound in G. jasminoides, its concentration in gardenia fruits, however, is rather low (about 0.005–0.01%), while geniposide presents abundantly (about 3.06–4.12%) [7]. It is difficult to extract genipin from gardenia fruits directly by common chemical procedures due to the instability of genipin. Acid hydrolyzation is not suitable because genipin reacts actively with acids [8]. On the contrary, genipin can be generated through microbial transformation in large quantity by ␤-glucosidase, which is produced during the fermentation process and is able to hydrolyze geniposide in gardenia fruits into

M. Xu et al. / Enzyme and Microbial Technology 42 (2008) 440–444

aglycone genipin [9]. However, the ␤-glucosidase is not special for hydrolyzing geniposide, so it’s not easy to transform geniposide into genipin completely, moreover the conditions of enzyme action are difficult to control. In our study, the fermentation of gardenia was conducted to transform geniposide into genipin by utilizing a fungal strain Penicillium nigricans isolated from our laboratory. The fermentation condition was optimized to maximize the production of genipin, and the isolation and purification of products were performed for its future application as a new biological and pharmaceutical material. 2. Methods 2.1. Preparation of gardenia extract Dry fruit of gardenia was finely ground to pass 200 meshes sieve and dried at 60 ◦ C before added to the treatment. Boil 100 g of gardenia with 1 L of water twice for 30 min at a time, and the extract was polled. The concentration of extract was adjusted to 1% by distilled water.

2.2. Strain screening The strain culture medium: potato dextrose agar (PDA) medium, to which 1% gardenia extract was added as the fermentation substrate before sterilization. The slab for strain screening: 5% gardenia extract, 1 mM glycin, and 1.8% agar powder. The mixture of soil and plants from the location, where gardenia plants grow well, was spread on the surface of the culture medium slab. The target strains were selected based on their activity of transforming geniposide determined by the size of blue circle on slab [10]. The strains screened were then inoculated on the fermentation culture medium and incubated at 30 ◦ C and 180 rpm for 4 days to harvest the crude enzyme. The measurement of activity of crude enzyme was performed by injecting 10 ␮l of the crude enzyme into the finestra on the strain screening slab with gardenia extract added and let the enzymatic transformation be undertaken at 50 ◦ C for 6 h. The size of the blue circle was measured and thus the enzyme activity could be determined from the circle size. Accordingly, the target strain with the highest enzymatic activity was gained. Spores of target strain were harvested for bran culture for 2 days and stored at 4 ◦ C until use.

2.3. Analysis of geniposide and genipin Thin layer chromatography (TLC) analysis of genipin, the fermentation product, was carried out on silica gel G TLC plate (20 cm × 10 cm) with mobile phase of the mixture of ethyl acetate and petroleum ether (2:1, v/v). The blue spot on the TLC was visualized by spraying glycine solution (0.44 mmol Gly dissolved in 0.1 M phosphate buffer at pH 7.0) and then heated at 70 ◦ C for 30 min. High performance liquid chromatography (HPLC, Shimadzu LC-10AT, Japan) analysis of both fermentation substrate and product was performed on a Reliasil C18 column (250 mm × 4.6 mm, 5 ␮m); the column temperature was maintained at 25 ◦ C. UV detection was set at 238 nm and the injection volume was 10 ␮l. The mobile phase used for analysis was methanol/water (45:55, v/v) at a flow rate of 1 ml/min. The linear regression equation for geniposide was Y = 2208.6x − 41.419 (R2 = 0.9999), and Y = 2478.4x + 14.149 (R2 = 0.9994) for genipin.

2.4. The optimization of fermentation conditions Spores of strain were inoculated into a 500 ml flask in which liquid media of tap water and the powder of gardenia were added, and then cultivated. The conditions of fermentation were established by one-factor-at-a-time method and an orthogonal test.

441

The influencing factors, including the content of gardenia, fermentation temperature, time, and rotation speed were optimized for the best established by an orthogonal layout L16 (45 ) in shake flask cultures.

2.5. Monitoring of gardenia fermentation process Fermentation was performed in a 10 L fermentor (GBRT) containing 7% gardenia extract at natural pH, 32 ◦ C, 300 rpm for 120 h, with inoculum volume of 5%. The aeration rate was 3.0 vvm. The amount of carbon source, nitrogen source, mycelial biomass, pH, enzyme activity, and the concentration of geniposide and genipin were measured at 4 h intervals. Mycelia were harvested at the end of fermentation, and then centrifuged at 10,000 × g for 10 min, before dried at 60 ◦ C in vacuum ovens to ensure constant weight. Total sugar was measured by phenol sulphuric acid method [11] and ammoniacal nitrogen by Kjeldahl method [12]. The activity of ␤-glucosidase was determined by pNPG method [13], while the concentration of geniposide and genipin by peak areas from HPLC analysis [14].

2.6. The extraction, separation and purification of genipin After fermentation, the broth was centrifuged at 10,000 rpm for 20 min to remove the insoluble compounds, and the supernatant was subjected to genipin extraction. Through the systemic all-over feedback control technique the separation of genipin by macroporous resin was optimized. Macroporous resins of varied aperture size, polarity and surface area were soaked in 95% ethanol for 24 h before being packed into the resin column. The resin was washed by 95% ethanol till the outflow was clear and mixed with distilled water in the ratio of 1:3 (v:v). Resins were then rinsed with distilled water till the outflow had no ethanol [15]. Using the systemic all-over feedback control technique [16], we evaluated the efficiency of five types of resins based on their absorption capacity and desorption rate to genipin. The measurement of adsorption capacity was performed by using the procedure as follows: mixture of 100 ml fermented broth and the resins prepared in the ratio of 10:1 were loaded onto 250 ml triangle bottles at 150 rpm for 24 h. The concentration of genipin was determined before and after the adsorption. Finally, the absorption capacity of macroporous resins was calculated according to the equation: Qe = (C0 − Ce )

Vi W

where Qe is the adsorption capacity at adsorption equilibrium (mg/g resin); C0 and Ce are the initial and equilibrium concentrations of genipin in the solutions, respectively (mg/ml); Vi is the volume of the initial feed solution (ml) and W the weight of the dry adsorbent (g). The measurement of desorption rate: wet macroporous resins that had adsorbed genipin were packed into the column and eluted with acetone–ethanol until no genipin flowed out, detected by HPLC. The concentration of genipin was determined in the 10 ml elute, and the following equation was used to quantify the desorption efficiency: D=

Cd Vd 100% (C0 − Ce )Vi

where D is the desorption rate (%); Cd is the concentration of genipin in the desorption solutions (mg/ml); Vd is the volume of the desorption solution; C0 , Ce and Vi are the same as those defined above. Adsorption of genipin was carried out in a glass column (2 cm × 30 cm) wetpacked with the selected resin. The bed volume (BV) of the resin was constant, and genipin in the eluent was monitored by HPLC analysis in the eluted aliquots collected at 5 ml intervals. When genipin was detected in the eluent indicated by TLC, the loading of the sample was stopped. The adsorbate-laden column was washed first with deionized water, followed by desorption with acetone–ethanol solution. The eluents were concentrated and dried under vacuum. The purification of genipin was carried out in a glass column (1 cm × 50 cm) packed with silica gel. Chromatography columns were packed as follows: the slurry of the silica gel (100 g) in petroleum ether (500 ml) was poured into a column half filled with petroleum ether. About 50 mg of the crude genipin

442

M. Xu et al. / Enzyme and Microbial Technology 42 (2008) 440–444

Fig. 2. HPLC analysis of gardenia extract pre-fermentation and after fermentation. (I) Standard geniposide solution; (II) standard genipin solution; (III) solution of gardenia extract pre-fermentation; (IV) solution of gardenia extract after fermentation.

3.2. The optimization of fermentation condition

Fig. 1. Process of genipin extraction and purification.

extract was dissolved in petroleum ether (10 ml) before loading on the silica gel column. The column was eluted with ethyl acetate–petroleum ether solution, and the eluates were collected with subsection mode. Pure genipin was obtained by recrystallization. The whole process of genipin extraction and purification was followed as shown in Fig. 1.

2.7. Structural identification of genipin Genipin obtained was identified by UV, MS, IR and NMR [17].

3. Results and discussion

By the one-factor-at-a-time method, the optimized cultural conditions were as follows: 10% (w/v) decoction of gardenia powder, rotation speed 180 rpm, temperature 30 ◦ C, medium volume 40%, and fermentation time for 120 h. The results of orthogonal test were outlined in Table 1. Based on the orthogonal test, the optimal conditions for submerged state fermentation were A2 B2 C3 D2 E2 , i.e. 10% of gardenia, at 30 ◦ C for 108 h, rotation speed 180 rpm, and 15% substance in volume. According to R-value, the volume of the medium had significant influence on the yield of fermentation product. The content of geniposide in gardenia was 4% before fermentation and 0.2% after fermentation, so the conversion rate of geniposide reached 95 %. The cost of the medium is a crucial factor in determining the feasibility of a fermentation process. Because there was no other nutrient added in the fermentation medium, it is expected that the production would be cost efficient.

3.1. Screening strains of high transformation activity 3.3. Characteristics of fermentation process Genipin can react with amino acids to form a blue pigment [18], thus microbial transformation of genipin can be evaluated by the size of the blue circle formed on the screening plate. The strain MK-1 with high microbial transformation capacity was screened easily and fast. This method has many advantages such as high selectivity, speediness and convenience. During the fermentation by MK-1, TLC results showed that sample I (pre-fermentation) did not show any blue spots while sample II (after fermentation) showed a clear blue spot with Rf 0.5. It indicated that substrate geniposide had been transformed into the product genipin. Four samples, including two standards of geniposide of 50 ␮g/ml (I) and genipin of 60 ␮g/ml (II), as well as the extract of 10 g/L from the mixture of pre-fermentation (III) and after fermentation (IV), were subjected to HPLC analysis. The retention time (Rt) of geniposide was 6.4 min, and genipin 10.2 min. After fermentation the absorption peak with Rt 6.4 min disappeared and a new peak with Rt 10.2 min appeared (Fig. 2). It was clear that the co-fermentation of MK-1 strain and gardenia converted geniposide into genipin.

According to the change of mycelial biomass during fermentation (Fig. 3), the growth of MK-1 was kept in the lag phase till 20 h of fermentation, while the residual sugar concentration (RSC) and ammoniacal nitrogen concentration (ANC) started to decrease sharply in this phase. During the fermentation period from 20 to 56 h, RSC and ANC decreased dramatically when the growth of MK-1 reached the exponential phase. At 56 h of fermentation, the growth of MK-1 reached the stationary phase. It was necessary to deplete energy sources in order to synthesize metabolic products, and this could be reflected by the increase of ANC but decrease of RSC. After 92 h of fermentation, both ANC and RSC declined slowly till the end of fermentation. The change of pH value was not remarkable during the entire fermentation process. The transformation of geniposide started at 52 h after inoculation, i.e. the stationary phase (Fig. 4.). During this period, the activity of ␤-glucosidases increased gradually till reaching the maximum value of 3.09 U/ml at 72 h, and the conversion rate increased along ␤-glucosidase activity. From 72 to 96 h,

M. Xu et al. / Enzyme and Microbial Technology 42 (2008) 440–444

443

Table 1 Partial results from orthogonal design L16 (45 ) in shake flask cultures Test

Gardenia concentration (%)

Temperature (◦ C)

Time (h)

Rotation speed (rpm)

Volume of medium (%)

Conversion rate (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 K1 K2 K3 K4 R

5 5 5 5 10 10 10 10 15 15 15 15 20 20 20 20 90.0 92.3 90.1 91.0 2.3

28 30 32 34 28 30 32 34 28 30 32 34 28 30 32 34 90.5 92.0 90.6 90.1 1.9

84 96 108 120 96 84 120 108 108 120 84 96 120 108 96 84 90.1 89.7 91.8 91.7 2.2

170 180 190 200 190 200 170 180 200 190 180 170 180 170 200 190 91.0 91.9 89.8 90.6 2.1

10 15 20 25 25 20 15 10 15 10 25 20 20 25 10 15 91.0 92.5 91.6 88.2 4.2

89.3 92.7 90.5 87.4 87.2 93.3 94.7 93.8 92.2 91.4 87.6 89.1 93.4 90.7 89.6 90.2

however, the conversion rate of geniposide decreased along with ␤-glucosidase activity. The concentration of genipin started stepping down as the fermentation process prolonged, probably due to the reaction between genipin and free amino acids. Therefore, the fermentation process should be terminated when geniposide is completely transformed and genipin should be extracted immediately to avoid the decrease in productivity. 3.4. The separation and purification of genipin The conditions for genipin extraction by the mixed macroporous resin of D141 and HPD100 (7:3, v/v) were optimized.

Fig. 3. The change of mycelial dry weight and the concentration of residual sugar and ammoniacal nitrogen during the fermentation by MK-1 in a 10-L batch fermentor.

The influencing factors and levels were described in Table 2. At pH 3, the solution flowed through the resin at the rate of 4 BV/h after the addition of 5% NaCl, with the sorption capacity of 23 mg/g. Yellow pigment and other impurities in the fermentation broth were washed off by distilled water first, and then by acetone–ethanol (3:2, v/v). The desorption rate was over 95%. The purity of genipin separated and purified by the resins above could reach 30% as determined by HPLC analysis. The mixture of partially purified genipin and the silica gel at 20:1 (w/w) was dried and grinded before mixed with petroleum ether at the rate of 1:10 (w/v). The mixture was then concentrated on the silica gel column and eluted by ethyl acetate/petroleum ether (3/2, v/v). The elute was then collected, vacuum concentrated to a small volume and purified again by repeating the procedure as mentioned above, till a high purity of genipin was obtained.

Fig. 4. The change of the concentration of geniposide and genipin with ␤glucosidases activity during fermentation.

444

M. Xu et al. / Enzyme and Microbial Technology 42 (2008) 440–444

Table 2 System action diagnostic test of genipin separation by macroporous resin Variable

Optimization result

X1

X2

X3

4 7 10

3.5 6 1

1.9 0.5 1.2

4

2

3

X4

X5

X6

X7

X8

X9

X10

X11

X12

Y1

Y2

10 0 5

5 50 95

95 50 5

2.2 0.5 3.9

0 2 4

0 2 4

2 4 0

2 4 0

8 0 4

18.9 8.1 4.7

92.7 12.2 17.8

5

40

60

2

0

0

0

3

7

23.2

96.7

Note: X1: pH of sample; X2: feed flow rate (BV/h); X3: concentration of sample (%); X4: concentration of salt (%); X5: concentration of ethanol (%); X6: concentration of acetone (%); X7: wash flow rate (BV/h); X8: wet weight of LSA-21 (g); X9: wet weight of HPD300 (g); X10: wet weight of D101-1 (g); X11: wet weight of HPD100 (g); X12: wet weight of D141 (g); Y1: sorption rate of macroporous resin; Y2: desorption rate of macroporous resin.

The product of high purity was distilled to be crystallized and re-crystallized. During crystallization, free amino acids from the broth were removed so that the reaction of product genipin and amino acids could be blocked and the loss of genipin would be avoided. Genipin of high purity was obtained by the combined application of macroporous resin and silica gel columniation. This procedure had the advantages including low environment pollution, speed, efficiency and low cost. 3.5. Identification of genipin The product purified by crystallization was identified by UV, MS, IS and NMR. The data were as follows: UV (CH3 OH) λmax 240 nm. ESI, m/z 226. IR υmax : 1007, 1105, 1686, 2849, 2929, 2935, 3030, 3238, and 3386 cm−1 . 1 H NMR (CDCl3 ) δ: 7.53 (s, H-3), 5.89 (s, H-7), 4.82 (d, J = 8.5 Hz, H-1), 4.35 (d, J = 13.2 Hz, H-10), 4.29 (d, J = 13.2 Hz, H-10), 3.74 (s, –OCH3 ), 3.22 (ddd, J = 9.5, 8.5, 8.5 Hz, H-5), 2.89 (ddt, J = 16.8, 8.5, 1.4 Hz, H-6), 2.54 (ddd, J = 8.5, 8.5, 1.5 Hz, H-9), 2.07 (ddt, J = 16.8, 9.5,1.8 Hz, H-6). The data above were in accordance with those of genipin reported before [17]. 4. Conclusions A strain of P. nigricans was obtained to transform geniposide in gardenia into genipin successfully and the conversion rate could reach 95% under the optimized fermentation conditions. No extra nutrient was needed in the fermentation medium, therefore the cost-efficient process has the potential to be applied on industry. Our study supplied new information for the optimization and development of microbial production of genipin from Gardenia jasminoides Ellis. Acknowledgements This work was financially supported by Sichuan Administration of Traditional Chinese Medicine of China (2004A34). The authors would like to thank Mr. Xingyu Zhang, Professor Wenquan Zou, and Professor Qi He for their technical assistance throughout this work.

References [1] Mie I, Satoshi M, Atsushi M, Kenji F. Japanese herbal medicine inchin-koto as a therapeutic drug for liver fibrosis. J Hepatol 2004;41:584–91. [2] Akao T, Kobayashi K, Aburada M. Enzymic studies on the animal and intestinal bacterial metabolism of geniposide. Biol Pharm Bull 1994;17:1573–6. [3] Zheng HZ, Dong ZH, Yu J. Modern research and application of Chinese Traditional Medicine, vol. 4. Beijing, China: Academy Press; 2000. p. 3166–72. [4] Koo HJ, Song YS, Kim HJ, Park EH. Antiinflammatory effects of genipin, an active principle of gardenia. Eur J Pharmacol 2004;495:201–8. [5] Chen YH. An in vivo evaluation of a biodegradable genipin-crosslinked gelatin peripheral nerve guide conduit material. Biomaterials 2005;26:3911–8. [6] Chen SC. A novel pH-sensitive hydrogel composed of N,O-carboxymethyl chitosan and alginate cross-linked by genipin for protein drug delivery. J Control Release 2004;96:285–300. [7] Cao JP, Wang YL, Jia YJ. Simultaneous determination of geniposide and genipin in Gardenia jasminoides Eillis by high performance liquid chromatography. J Dalian Med Univ 2001;23:61–2. [8] Yao XS, Wu LJ, Wu JZ. Natural medicine chemistry, vol. 220. 4th ed. Beijing, China: People’s Medicinal Publishing House; 2004. [9] Shigeaki F, Shigeyuki N, Kunimasa K, Jun-Ichi K. Continuous blue pigment formation by gardenia fruit using immobilized growing cells. J Ferment Technol 1987;65:711–5. [10] Paik YS, Lee CM, Cho MH, Hahn TR. Physical stability of the blue pigments formed from geniposide of gardenia fruits: effects of pH, temperature, and light. J Agric Food Chem 2001;49:430–2. [11] Xiao JH, Chena DX, Xiao Y, Liu JW, Liu ZL, Wan WH, et al. Optimization of submerged culture conditions for mycelial polysaccharide production in Cordyceps pruinosa. Process Biochem 2004;39:2241–7. [12] Page AL, Miller RH, Keeney DR. Method of soil analysis. Part 2. Chemical and microbiological properties, No. 9. 2nd ed. Madison, WI, USA: ASA, SSSA Publishing; 1982. p. 513–7. [13] Kuo LC, Cheng WY, Wu RY, Huang CJ, Lee FK.T. Hydrolysis of black soybean isoflavone glycosides by Bacillus subtilis natto. Biotechnol Prod Process Eng 2006;73:314–20. [14] Ueno K, Lim JM, Bhoo SH, Paik YS, Hahn TR. Simultaneous estimation of geniposide and genipin in mouse plasma using high-performance liquid chromatography. Anal Sci 2001;17:1237–9. [15] Wang DM, Wei Q, Ma XH. Application of macroporous on the isolation of the constituents in pharmaceutical plants. J Northwest Forestry Univ 2002;17:60–3. [16] Chen YZH, Sun QL. The research of effective method for the mutation breeding of a bacterium with high ␥-poly (glutamic acid) productivity. J Sichuan Univ (Nat Sci Edition) 2005;42:389–94. [17] Endo T, Taguchi H. The constituents of Gardenia jasminoides: geniposide and genipin-gentiobioside. Chem Pharm Bull 1973;21:2684–8. [18] Lee SW, Lim JM, Bhoo SH, Paik YS, Hahn TR. Colorimetric determination of amino acids using genipin from Gardenia jasminoides. Anal Chim Acta 2003;480:267–74.