search
for
 About Bioline  All Journals  Testimonials  Membership  News


African Journal of Traditional, Complementary and Alternative Medicines
African Ethnomedicines Network
ISSN: 0189-6016
Vol. 4, Num. 2, 2007, pp. 157-164

African Journal of Traditional, Complimentary and Alternative Medicines, Vol.4, No. 2, 2007, pg. 157-164

 Research Paper 

BIOLOGICAL ACTIVITIES OF SCHEFFLERA  LEUCANTHA                   

Buppachart Potduang, Chanchai Chongsiriroeg, Yaowaluck Benmart, Rattanasiri Giwanon, Winai Supatanakul and Sayan Tanpanich

Pharmaceuticals and Natural Products Department Thailand Institute of Scientific and Technological Research, Technopolis, Thanon Liab Klong 5, Klong Luang, Pathumthani 12120, Thailand. 
E-mail: buppachart@hotmail.com; buppachart@tistr.or.th
Phone: 66 2577 9108     Fax: 66 2577 9108  

Code Number: tc07030

Abstract

This study investigated various biological activities of the ethanolic extract of dried ground leaves of Schefflera leucantha Viguier(Araliaceae).    The extract possessed very low cytotoxicity to brine-shrimp with the LC50 of 4,111.15mg/ml; the significant antioxidant activity on DPPH with the EC50 of 71.90 μg/ml; the inhibitory activity on mushroom tyrosinase with the IC50 of 10.53mg/ml using the dopachrome microplate-assay.  The extract of 5-20mg/ml range in the agar dilution assay were active against various pathogenic microbial (11 species, 11 strains), with the minimum inhibitory concentration (MIC) of 5mg/ml against Clostridium spp.; MIC=10mg/ml against enteropathogens as Bacteroides spp., Enterococcus faecalis ATCC 29212, Lactobacillus spp.,Peptococcus spp. and Streptococcus mutans; MIC=10mg/ml against a pneumonia causing bacteria Klebsiella pneumoniae and a dermatopathogen as Propionibacterium acnes; MIC=20mg/ml against dermatopathogens as Staphylococcus aureus ATCC 6538, Streptococcus spp. and Candida albicans ATCC 90028.  TLC fingerprints of the specific extracts from the leaf powder exhibited zones of steroids-terpenes and flavonoids.  HPLC fingerprint of the flavonoid extract was performed.   

Key words:Schefflera leucantha, Antioxidant, Antityrosinase, Antimicrobial activity, Phytochemistry             

Introduction 

Schefflera leucanthaViguier, ARALIACEAE, locally known in Thailand as ‘Hanuman Prasankai’ is a 1-2m shrub, highly branched,  cultivatedelsewhere in the moist open field.   The finger-like leaf composed of 5-6 compound leaves, oblanceolate-subulate,glabrous, 1.5-2cm broad, 5-10 cm long, with a 3-9cm long petiole.  The white flowers are small, inflorescence.  Theyellow fruits are dense aggregate.  The infusion of this plant is said to relieve a cold, allergies, asthma, chronic coughand respiratory tract infection.  The whole plant is also used as analgesics, antipeptic and to promote bloodcirculation.  The leaves are used for wound-healing, inflammation, cough, bronchitis, carcinogenic and removal ofblood clot in the brain. 

In Thailand and China the plant is widely used as an antiasthmatic.  It was reported that the active bronchodilator principle is a mixture of saponins (Pancharoen et al., 1994), which showed no sign of toxicity at the dose of 1000mg/kg orally (Witthawaskul et al.,2003).  Aqueous extract of S. leucantha has been reported having hypoglycemic activity in  rats (Satayavivad et al., 1996).

In the present study, we investigated various biological activities of the crude ethanol leaf extract of S. leucantha to provide new fields for the utilization study of the plant.  TLC fingerprints and HPLC fingerprint of the leaf powder were performed for future references.

Materials and Methods

Plant material 

Provided fresh leaves of S. leucantha were collected in November 2003 from Nakhon Rachasima province, Thailand.  The plant was identified and authenticated by the Research Officer (Botany).  Thesample was dried in a hot air oven at 40-50oC, and then pulverized into powder.  The authentic plant harbarium of voucher specimen no. LRS-0111 has been kept at the Lamtakhong Plant Research Station,TISTR, where the plant are also cultivated.

Preparation of crude ethanol extract

The leaf powder was 3 times repeatedly macerated with fresh 95%ethanol in a percolator, then filtered through the cotton plug.  The combined filtrate was evaporated to dryness under reduced pressure at 40-50°C.  The resulting crude ethanol extract was then stored at 4oC until used.

Cytotoxicity to brine-shrimp 

Modified method of Solis et al. (1992) was used to determine the inhibitory activity on Artemia sp. in a 96-well micro-plate.  100 μl of the crude ethanol extract solution in 0.25%Tween80-artificial seawater was added into each well containing 5 newly hatched brine shrimps in 100 μl artificial seawater, then incubated at room temperature for 24h.  All samples were repeated in 6 wells to make overall tested organisms of 30 for each.  Counted the dead organisms under a binocular microscope (4x).  Plot %Lethality vs Log concentration.  Substituted y = 50 in the resulted linear equation to obtain the x value.  The antilog x was then the LC50 (conc. of 50%lethality) value (Ballantyne et al., 1995).  Thymol and kojic acid were used as reference standards.   

Antioxidant activity

Scavenging of DPPH radicalModified method of Hatano et al. (1989) was used to measure the free radical scavenging activity of the crude ethanol extract on DPPH (2,2-diphenyl-1-picrylhydrazyl from Sigma, Germany) which is a stable radical.  In a 96-well micro-plate, added 100ml of the extract solutions (in absolute ethanol) into each well containing 100 μl of 0.06mM DPPH in ethanol,mixed well, and measured the absorbance at 517nm exactly at 30min by a micro-plate reader (TECAN, Sunrise remote).  All samples were run in triplicate. Determined %scavenging of test samples as follows:

      

Where A, B and C represent the absorbances of DPPH in the resulted reactionmixture, the blank, and the control respectively.  Plot %Scavenging vs Log concentration.  Substituted y = 50in the resulted linear equation to obtain the x value.  The antilog x was then the EC50(conc. of 50%scavenging)value (Ballantyne et al., 1995).  BHT, BHA and vitamin C were used as reference standards.    

Anti-tyrosinase assay 

20%ethanol extract derived from the crude ethanol extract, was assayed for enzyme tyrosinase inhibition by the dopachrome method modified fromIida et al. (1995),  using L-Dopa (Sigma Chemical) as a substrate.   In a 96-well micro-plate, mixed 150 μl of 0.02M sodium phostphate buffer (pH 6.8) with 50μl of the sample solution and 50ml of mushroom tyrosinase solution (314.8U/ml, Fluka).   Pre-incubated the mixture at 25oC for 10min before adding 50ml of 0.34mM L-Dopa, and then incubated at 25oC for another 2min.  Measured the absorbance at 492nm of the biosynthesized red dopachrome by a micro-plate reader (TECAN, Sunrise remote).  All samples were run in triplicate.  The absorbance differences before and after the incubation were used to calculate the percentage inhibition of tyrosinase as follows: 

Where the absorbance difference A represents the control (L-Dopa mixed with enzyme in buffer); B represents the blank (L-Dopa in buffer); C represents the reaction mixture; and D represents the blank of C (L-Dopa mixed with test sample in buffer).  Plot %Tyrosinase inhibition vs Log concentration.  Substituted y = 50 in the resulted linear equation to obtain the x value.  The antilog x was then the IC50 (conc. of 50%inhibition) value (Ballantyne et al., 1995).  Kojic acid, a well-known tyrosinase inhibitor, was used as the reference standard.

Anti-microbial assay

The activities against various pathogenic microorganisms were screened using the agar dilution method (Washington and Sutter, 1980).  The test microorganisms were maintained on specific assay media as Mueller Hinton Agar (MHA; Difco Laboratories) for aerobes; WC Agar (Wilkins and Chalgren, 1976) for anaerobes; and Saboraud Dextrose Agar (SDA; Difco Laboratories) for yeasts.  Inoculums derived from the cultures were incubated at 37oC in corresponding broths (18-24h for aerobes in MHB; 48h for anaerobes in WC Broth; 48h for yeast in SDB).  A suspension of each isolates was prepared in a fresh inoculum’s medium and adjusted to McFarland 0.5 turbidity standard.  Prepared dilution plates (0.5-20mg/ml) of the crude ethanol extract by adding appropriate volumes of the stock solution (0.8g/ml in acetone) into the assay medium.  Spot inoculated the dilution plates with the isolates suspensions, and then incubated at 37oC (overnight for aerobes; 3 days for anaerobes; 48h for yeast).  Minimum inhibitory concentrations (MICs) of the extract were determined.

TLC fingerprints 

Thin-layer chromatography (TLC) of 2 different extracts containing either steroids-terpenes or flavonoids from the leaf powder were performed on 0.25mm thick TLC plates (Merck Silica gel 60 F254-precoated) using suitable developing solvent systems, and special detection reagents (Merck, 1980; Wagner and Bladt, 1996).  No alkaloid could be extracted from the leaf powder.  These chemical groups were chosen to perform the reference TLC fingerprints because they have been the easily extracted groups possessing various bilogical activities.

1. TLC fingerprint of the steroids and terpenes.  The steroids-terpenes extract was prepared by stirring 1g of the leaf powder with hexane (3x50ml) for 30min, filtered, evaporated the filtrate to dryness under reduced pressure, and then dissolved in 1ml chloroform.  Applied 3μl of the extract onto a TLC plate and perform chromatography with a suitable solvent system to a distance of 10cm.  Sprayed the developed plate with vanillin-sulfuric acid reagent, then heated until the spots attain maximum colour intensity of the existingsteroids-terpenes compared to ref. std. β -sitosterol (Sigma, USA) in chloroform (1:1 w/v).   Figured the chromatogram by a scanner.           

2. TLC fingerprint of the flavonoids.  The flavonoids extract was prepared by stirring 0.5g of the leaf powder with 5ml methanol on a dry block heat bath (60°C, 5min), allowed to cool, filtered, evaporated the filtrate to dryness under reduced pressure.  Dissolved the dried extract in 1ml methanol, then applied 3μl onto a TLC plate and perform chromatography to 10cm with a suitable solvent system.  Sprayed the developed plate with natural products-polyethylene glycol (NP/PEG) reagent, and detected fluorescing zones of the existing flavonoids under UV-365nm compared to ref. std. rutin (Fluka, Switzerland) in methanol (1:1 w/v).  Figured the UV-365nm chromatogram by a digital camera (Keter KT-1000F, Taiwan).                       

HPLC fingerprint

A methanolic extract containing flavonoids was prepared by shaking1g of the leaf powder with 50ml of methanol at 1,500rpm for 2min, filtered through a Whatman paper no.41 into a volumetric flask and made up to 50ml with methanol.  Filtered the extract through a 0.45μ nylon syringe filter membrane before subjected to binary gradient RP-18, 30°C, 1ml/min flow rate, HPLC analysis with 270nm UV detector.  Solvent A was water with 0.2%TFA+10%methanol, and solvent B was acetronitrile with 0.3%TFA.  Standard addition of rutin (Merck, Germany) and quercetin (Fluka, Switzerland) was applied to HPLC chromatogram.

Results

The extraction process yielded 9.01% of the crude ethanol leaf extract of S. leucantha. The biological activities of theextractexhibited the computerized graphical calculated LC50 of 4,111.15mg/ml on brine shrimp cytotoxicity, EC50 of 71.90 μg/ml on DPPH radical scavenging.  The 20%ethanol extract derived from the crude extract gave the IC50 of 10.53mg/ml on mushroom tyrosinase inhibition (Tables 1, 2, 3, 4).

The agar dilution method assay indicated that the crude extract was active against various representative disease causing microorganisms (11 species, 11 strains) within the concentration range of 5-20mg/ml.  The minimum inhibitory concentrations (MICs) were 5mg/ml against Clostridium spp.; 10mg/ml against enteropathogens as Bacteroides spp., Enterococcus faecalis ATCC 29212, Lactobacillus spp.,Peptococcus spp. andStreptococcus mutans; 10mg/ml against a pneumonia causing bacteria Klebsiella pneumoniae and a dermatopathogen as Propionibacterium acnes; and 20mg/ml against dermatopathogens as Staphylococcus aureus ATCC 6538, Streptococcus spp. and Candida albicans   ATCC 90028 (Table 5). 

TLC fingerprints of the specific extracts fromS. leucanthaleaf presented a specific pattern of steroids-terpenes developed in dichloromethane-ethylacetate-formic acid (60:5:1); and a specific pattern of flavonoids developed in ethyl acetate-formic acid-acetic acid-water (100:11:11:26) as shown in Table 6, Figure 1.

RP-HPLC fingerprint of the flavonoid extract fromS. leucanthaleaf, under a suitable 40min-program linear gradient at 270nm, was shown in Figure 2.

Discussion

The concentration of 50%activity of the S. leucantha leaf extracts were calculated from the computerized linear equations as followed: y = 164x − 542.69 on brine shrimp cytotoxicity; y = 40.784x − 25.659 on DPPH radical scavenging; and y = 164x − 542.69 on tyrosinase inhibition.  Where x was obtained by substituting y = 50, the antilog x gave the value of either the LC50, EC50 or IC50, respectively.   The mild brine shrimp inhibition could be correlated with the traditional uses of squeezed extract of fresh leaves against skin cancer, breast cancer and cancer of the cervix.  Further study against cancer cell lines should be performed.

The DPPH radical scavenging activity (approx.17.08, 17 and 58.94 times less effective than BHT, BHA and vitamin C respectively), suggested the leaves as a source of antioxidants and be effective in diseases caused by overproduction of radicals.

The anti-tyrosinase activity (approx.4578 times less effective than standard kojic acid) meant there were some melanin biosynthesis inhibitors in the 20%ethanol extract.

 The in vitro anti-microbial activity against various pathogens coincided with the traditional uses to relieve respiratory tract infection.

Zoning patterns of steroids-terpenes and flavonoids on the TLC fingerprints, as well as the RP-HPLC fingerprint of the flavonoid extract, were specific enough to be references for future identification of the S. leucantha leaf powder. 

Conclusion

These results revealed that the leaves of S. leucantha had some values for further utilization study, either to be developed as an anti-cancer or a source of antioxidants, anti-hyperpigmentation agents, and antibiotics.

Acknowledgements           

We thank the Pharmaceuticals and Natural Products Department Thailand Institute of Scientific and Technological Research (TISTR) for providing the fund and good laboratory facilities, and to Mr. Parinya Wilairatana, Ex-Director of the Lamtakhong Plant Research Station TISTR for providing the plant materials.

References

  1. Ballantyne, B., Marrs, T. and Turner, P.  (1995). General & applied toxicology.  A bridged ed.  London : Macmillan press.
  2. Hatano, T., Edamatsu, R., Hiramatsu, M., Mori, A., Fugita, Y., Yasuhara, T., Yoshida, T. and Okuda, T. (1989).  Effect of the interaction of tannin with co-existing substance VI. Effect of tannins and related poly-phenols on superoxide anion radical, and on 1,1-diphenyl-2-picrylhydrazyl radical.  Chem. Pharm.  Bull. 37: 2016-2012.
  3. Iida, K., Hase, K., Shimomura, K., Sudo, S., Katota, S. and Namba, T.  (1995).  Potent inhibitors of tyrosinase activity and melanin biosynthesis from Rheum officinale.  Planta Med.  61: 425-428.
  4. Merck, E.(1980). Dyeing reagents for thin-layer and paper chromatography.E. Merck, Damstadt.
  5. Pancharoen, O., Tuntiwachwuttikul, P., Taylor, W.C., Picker, K. (1994). Triterpenoid glycosides from Schefflera lucantha.  Phytochemistry 35(4): 987-92.
  6. Satayavivad, J.,Bunyapraphatsara, N., Thiantanawat, A.. and Kositchaiwat, U. (1996).  Hypogly-cemic activity of the aqueous extract of Schefflera  leucantha Viguier in  rats.  Thai Journal of Phytopharmacy 3 (1): 1-5.
  7. Solis, P.N., Wright, C.W., Anderson, M.M., Gupta, M.P. and Phillipson, J.D.  (1992). Amicrowell cytotoxicity assay using Artemia salina (Brine Shrimp).  Planta Med. 59: 250-252.
  8. Wagner, H. and Bladt, S.  (1996).  Plant Drug Analysis.  Springer-Verlag, Berlin.
  9. Washington II, J.A., and Sutter, V.L.  (1980).  Dilution susceptibility test: agar and macro-broth dilution procedure.  In: Mannual of Clinical Microbiology.  3rd ed.  American Society for Microbiology, Washington D.C. 
  10. Wilkins, T.D. and Chalgren, S.  (1976).  Medium for use in antibiotic susceptibility testing of anaerobic bacteria.  Antimicrobial Agents and Chemotherapy Dec. 10(6):9265-28.
  11. Witthawaskul, P., Panthong, A., Kanjanapothi, D., Taesothikul, T., Lertprasertsuke, N.  (2003).  Acute and subacute toxicities of the saponin mixture isolated from Schefflera leucantha Viguier.   J. Ethnopharmacol. 89(1):115-121.

© Copyright 2007 - African Journal of Traditional, Complementary and Alternative Medicines


The following images related to this document are available:

Photo images

[tc07030t5.jpg] [tc07030t4.jpg] [tc07030f1.jpg] [tc07030t3.jpg] [tc07030f2.jpg] [tc07030t6.jpg] [tc07030t1.jpg] [tc07030t2.jpg]
Home Faq Resources Email Bioline
© Bioline International, 1989 - 2024, Site last up-dated on 01-Sep-2022.
Site created and maintained by the Reference Center on Environmental Information, CRIA, Brazil
System hosted by the Google Cloud Platform, GCP, Brazil