Pergamon 0031-9422(94)tKM4-1 Phyrochemaswy. Vol. 31. No 5. pp 1273- 1276. 1994

Copyright Q 1994 Elwvier Science Ltd Pnntcd I” Great LIntam All nghts reserved

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COMPARATIVE POLYPHENOLIC PRODUCTIONS IN CRATAEGUS MONOGYNA CALLUS CULTURES

T. BAHORUN, F. TROTIN* and J. VASSEUR~

Laboratoire de Physiologic Cellulaire et Morphogenke Vtgktale, U.S.T.L., I.N.R.A., Universitt des Sciences et Technologies de Lille, 59655 Villeneuve d’Ascq Cedex, France, l Laboratoire de Pharmacognosie, Facultk des Sciences Pharmaceutiques et Biologiques, B.P

83, 59006 Lille Cedex, France

(Received in revised form 17 May 1994)

Key Word Index-Crotaegus monogyna; Rosaceae; proanthocyanidins; catechins; phenolic acid; flavonoids; callus culture.

Abstract-The production of polyphenols by two-year-old callus cultures from floral buds of Crataegus monogyna has been studied in relation to growth variation within a subculture period. Calli produce mainly proanthocyanidins (1074-2959 mg 100 g-‘dry wt), B2 dimer (86-321 mg 100 g-‘dry wt), ( - )-epicatechin (468.-807 mg 100 g “dry wt), chlorogenic acid (213-769 mg 100 g- ‘dry wt), and a major flavonoid hyperoside (61-143 mg 100 g- ‘dry wt). Optimum production of polyphenols seems to be generally attained when maximal growth is reached.

INTRODUCIION

Hawthorn and its extracts are commonly used in phar- maceuticals owing to their sedative [ 11, vasculoprotective and coronary blood vessel flow enhancing properties [2]. These effects have been mainly attributed to flavonoids and proanthocyanidins [ 11. Plant phenolics including proanthocyanidins, catechins, flavonoids and phenolic acids, are known to exhibit antioxidant and free radical scavenging properties [3-S]. In a previous work, inter- esting antilipoperoxidant activities of fresh hawthorn plant extracts were shown to be related to these classes of substances [6]. As part of a program concerning the phenolic production of cell cultures [7-93, calli were

initiated from flower buds of Cratoegus monogyna Jacq. Here, we report the comparative synthesis of proantho- cyanidins including the B2 dimer, catechins, anthocyan- ins, chlorogenic acid and a major flavonoid, hyperoside, as well as a low yield of isoquercitrin, with respect to mass increase, from these cultures.

RESULTS AND DISCUSSION

Callus cultures initiated from flower bud ovaries were maintained over a period exceeding two years. They were characterized by an initial low yield of polyphenols (weeks O-27) followed by a progressive enhancement and finally a stabilization in the production. To study the optimal period of different polyphenol synthesis in rela- tion with growth, stabilized callus cultures were analysed within a culture period of 40 days with a four day analysis

+Author to whom correspondence should be addressed.

interval after subculturing 21-day-old calli which repres- ent the day 0 values shown on the graphs. Figure 1 depicts the total phenolic content in relation to growth. Optimum growth increase is observed from days 24 to 32 (177- 179 mg 100 g- ’ dry wt). During that time, maximal production of total phenols appeared at day 28 (5890 mg 100 g- I dry wt). The initial growth period (ca days 4-16) showed an intense synthesis of total phenols with, however, a low biomass production.

Optimal production is observed between the 24th and 32nd day of culture for global proanthocyanidins (GPC) (2750-2950 mg 100 g- ’ dry wt), oligomeric proantho-

cyanidins (OPC) (IllO-1020mglOOg-’ dry wt) and polymeric proanthocyanidins (PPC) 1300- 1720 mg 100 g-l dry wt) (Fig. 1). Anthocyanin levels (Fig. 1) remained relatively constant from days 12 to 24 (189-163 mg 100 g- ‘dry wt) then increased to maximum production at days 28 and 32 with 266 and 230 mg 100 g- ’ dry wt, respectively.

The analysis of individual compounds, shows an in- crease of (-)-epicatechin from day 4 to day 16 (468-807 mg 100 g-‘dry wt) followed by a maximum production from days 24 to 28 (807 and 838 mg 100 g- ’ dry wt, respectively) Proanthocyanidin B2 vari-

ations seem analogous with those of total and oligomeric fractions and are related to (- tepicatechin production. A rapid increase from day 4 to 16 (from 86 to 262 mg 100 g- * dry wt) is followed by a maximum yield for days 24 and 32 (321 and 290 mg lOOg-’ drywt, respectively). In both cases, a decrease is recorded on day 20 [( - )-epicatechin: 542 mg 100 g ’ dry wt and B2 dimer: 60 mg 100 g- ’ dry wt]. This correlation between B2 dimer and ( - )-epicatechin production is consistent

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1274 T. BAHORUN et al.

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Fig. I. Total phenols ( q ), total proanthocyanidin (GPC (W)), oligomeric proanthocyanidin (OPC (H)), polymeric

proanthocyanidin (PPC (M)) and anthocyanidin (3) yields by C. monog~na callus cultures versus growth increase (--X-l

with literature data [IO] and our observations concer- ning epicatechin galloyl esters in Fagopyrum esculentum cultures [9] showing that (-)-epicatechin-rich plant or- gans generally contain proanthocyanidin B2 as major dimer.

The main phenolic acid, chlorogenic acid increased from day 4 to 20 (213 349 mg lOOg_’ drywt) followed by a peak production at day 24 (769 mg 100 g- ’ dry wt). Hyperoside, the major flavonoid, (quercetin 3-galacto- side), varied little. the amount remaining within

61- 143 mg 100 g ’ dry wt, respectively, on days 4 and 36. Another flavonoid. isoquercitrin (quercetin 3-glucoside), has been detected in trace amounts (mean values: 20 30 mg 100 g- ’ dry wt). Our cultures thus provide a simpler phenolic qualitative pattern when compared to that of plant organs, particularly floral buds [6, 111.

Our data concerning antioxidant activities of fresh hawthorn extracts indicate that the most efficient antili- poperoxidant compounds [6] were in decreasing order

B2 dimer, ( - )-epicatechin, hyperoside and chlorogenic acid while polymeric proanthocyanidins lacked any ac- tivity. The studied calli synthesize appreciable amounts of the most active compounds with maximal yields related to the optimum growth stages.

For comparison, some of the numerous literature data dealing with in citro polyphenol production must be cited. In the held of anthocyanins, similar contents were obtained in buckwheat calli (12) but values as high as 13% dry wt have been reported in Arabia cordata cell cultures (13). The flavonoid yield in our hawthorn calli is moderate and practically restricted to only one derivat- ive. Flavonols not found in the original plant have been isolated in Vancouueria hexandra (14) and significant amounts of flavones up to 9.07 % dry wt have also been reported in Scutellaria haicalensis (15). As far as pro- anthocyanidins and catechins are concerned the greatest

yields reported for total proanthocyanidins, including B dimers, are those from Ginkgo hiloba (up to 600 mgg- ’ dry wt) and Pseudotsuga menziesii cultures [16]. Tissue cultures of Uncaria elliptica synthesize 520 mg 100 g - ’ dry wt of ( - )-epicatechin (17) As shown, our callus cultures produce noticeable amounts of pro- anthocyanidins and B2 dimer between days 24 and 32. In a study concerning buckwheat calli we observed the optimal synthesis for B2 and BZ-3-O-gallate dimers at a similar period (180 and 600 mg 100 g- ’ dry wt, respect- ively) [9].

In the case of C. monogyna, studies concerning calli and cell suspensions initiated from other plant parts under different conditions have been made. (-)-Epicatechin, (+)-catechin and oligomeric proanthocyanidins have been reported in cell suspension while chlorogenic acid and different hydroxycinnamic and benzoic acids were present in callus cultures [18]. More recently [19], cell cultures from shoot tips, analysed for a period of six months showed a more complex and interesting flavon- oid pattern with the isolation of vitexin, vitexin-2”-O- rhamnoside, rutin and hyperoside, the latter being domi- nant (I .86- 12.58 mg 100 g - * dry wt). Total proantho- cyanidins varied from 4 to 17 mg 100 g- ’ dry wt.

EXPERIMENTAL

Tissue culture

Callus cultures were initiated from hawthorn floral buds deprived of sepals, petals, anthers, stigmas and pistils harvested in May 1991 in the region of Valencien- nes (north of France). Culture was made in sterile Petri dishes (90 mm diameter) containing 25 ml nutrient medium solidified by 0.5% agar (Biokar type E, Prolabo

Polyphenols in Crataegus monogyna 1275

France). The culture medium contained B, Gamborg (20) mineral soln elements, sucrose (30 g I - ‘), casein hydrolys- ate (100 mg l- I), myoinositol (100 mg I- I), pyridoxine (0.5 mg I- I), nicotinic acid (0.5 mg I- ‘), thiamine (0.5 mgl- ‘), kinetin (0.5 mgl-‘) and 2,4 D (2 mgl- ‘). The pH was adjusted to 5.6 before autoclaving (1 lo” for 20 min). Calli were initiated under 16: 8 light/dark period at 22 + 2” and after an induction period (9 subcultures), transferred to permanent light (50 pmol mm2 set- ‘) at the same temp. Subcultures were performed every 3 weeks (4 calli Petri dish - ‘, each 125- 150 mg fr. wt). Two-year- old stabilized callus cultures producing relatively con- stant amounts of phenolics were harvested every 4 days for 40 days, each analysis being carried out at least 3 times to give mean values and s.d. Voucher specimens have been deposited at the Department of Pharmacognosy of the Faculty of Pharmacy, University of Lille II.

Extraction

Fresh tissues (10 g) were extracted with MeOH-Hz0 (7:3) (2 x 100 ml), Me&O (7:3) (2 x 100 ml) and finally with MeOH 100% (2 x 100 ml). Filtrates were coned and the resulting aq. extract divided into 2 parts. Part 1, corresponding to 5 g fr. wt was used to obtain the following EtOAc extracts (see below). Part 2 (5 g fr. wt) was carefully evapd to dryness and the residue dissolved in absolute MeOH for storage at - 20”, this stock soln (extract I) being adjusted at a final l/5 (plant fr. wt vol.) ratio. The Part I H,O extract was partitioned with EtOAc (5 x 100 ml). EtOAc, dehydrated layers were ev- apd to dryness and dissolved in absolute MeOH to yield a l/5 (plant fr. wt vol.) ratio (extract II). The remaining aq. phase following EtOAc extraction was evapd as above and dissolved in absolute MeOH to obtain l/5 (plant fr. wt vol.) ratio (extract III).

TLC analysis

TLC of extracts I, II, and III was in the following systems: ID-TLC (Silicagel, Merck) Toluene-Me,CO- HCOOH, 3:3:1 for proanthocyanidins [21], C,H,- Me&O, 4: 1 for proanthocyanidin peracetates [22], EtOAc-HCOOH-H,O, 8: 1: 1 for flavonoids [ll]. 2D- TLC on cellulose plates (Merck) (solvent 1: 6% HOAc in H,O, solvent 2:2-BuOH-HOAc-H,O, 14:1:5 for pro- anthocyanidins [lo]. Proanthocyanidins were visualized by anisaldehyde-H,SO, spray reagent while flavonoids were revealed by 2-aminoethyldiphenylborate reagent followed by 5% polyethyleneglycol 4000 in absolute EtOH.

Calorimetric analysis

Determination of total phenol content. Total phenol estimation was determined by the method adapted from Singleton et al. [23] at 680 nm using the Folin-Ciocalteu reagent. Results were expressed in mg of gallic acid 100 g-’ of dry wt.

Determination of anthocyanin content. Anthocyanin content was expressed in mg cyanidin chloride 100 g- 1 dry wt after recording the extinctions of meth- anolic extracts in BuOH-cont. HCI (19: 1) at 550 nm.

Determination oJ proanthocyanidin content. Proantho- cyanidin contents were assayed by the standardized method of Porter et al. [24]. To each tube is added 0.5 ml of methanolic extract, 6 ml of a soln of BuOH-cont. HCl (95: 5) and 0.2 ml of 2% (w/v) solution of NH,Fe(SO,,), lZH,O in 2M HCI. The tubes were capped, thoroughly mixed and heated 40 min in a constant level H,O bath run at 95f0.2”. Solns were cooled and extinctions re- corded at 550 nm. The amount of pranthocyanidin was expressed in mg of cyanidin chloride 100 g- ‘of dry wt.

HPLC analysis. HPLC analysis was carried out after filtration of methanolic extracts on Millipore (0.45 pm) and injection (20 ~1) on a Ultrasphere RP18 (5 pm) re- versed phase Cis column (46 mm i.d. x 150 mm) by an acetonitrile-H,O gradient [9]. UV detection: 280 nm for proanthocyanidins and phenolic acids, 360 nm for flav- onoids. ( - )-Epicatechin, chlorogenic acid, hyperoside and isoquercitrin were identified and quantified by com- parison with authentic samples (Extrasynthbe). B2 dimer, obtained from F. esculentum cell cultures [9] was also used for quantitation.

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