ABSTRACT-
Medinilla septentrionalis is shrub, wildly distributed in Nui Ba National park, Lam Dong, Vietnam. Although there is no scientific publication about the Medinilla septentrionalis as medicinal plant, the plant has been used for diarrhea treatment by ethnic minorities there. In this study M. septentrionalis ethanolic extract was used to evaluate antibacterial activity, toxicity and anti-diarrheal activity. The results showed that M. septentrionalis ethanolic extract had highly antibacterial activity, particularly to diarrhea relating bacteria such as Salmonella spp., Shigella spp., Vibrio spp. and Escherichia coli. The M. septentrionalis ethanolic extract also effectively prevented enteropooling, reduced either time of charcoal transit in small intestine or defecation in castor oil-induced mice at 63 mg kg–1 body weight. In addition, no significant toxicity signs and mortality were observed on mice after treating the plant extract up to doses of 10000 mg kg–1 body weight. The preliminary phytochemical screening of M. septentrionalis ethanolic extract showed the presence of carbohydrates, saponins, cardiac glycosides, flavonoids, phenolic compounds, tannins and steroids. These results strongly demonstrated that M. septentrionalis ethanolic extract possessed highly anti-bacterial and anti-diarrheal properties. The results in this study contributed a validation data for the use of M. septentrionalis in diarrheal treatment.
Key-Words- Medinilla septentrionalis, Antibacterial activity, Anti-diarrheal activity, Ethanolic extract
INTRODUCTION-
Diarrhea is one of the popular diseases which leading causes of death among children under five globally, especially in the developing countries. Every year, there are more than 5-8 million deaths all over the world [1]. Diarrhea is caused by many reasons including bacterial infections such as Salmonella enteritidis, Salmonella typhii, Shigella flexneri, Escherichia coli, Vibrio cholerae, Clostridium difficile or chemicals such as castor oil or magnesium sulfate [2-3]. These agents cause the influx of water and ions to the lumen and thus increase the intestinal motility, thereby using watery stool [4].
To treat diarrhea, some medicine are used such as
diphenoxylate, loperamide. However, the usage of those
medicines may cause some side effects as vomit, intestine
obstruction and constipation [5]. For this reason, recently
there has been great interest in herbal remedies for diarrhea
treatment with no side effects.
Medinilla septentrionalis (Melastomataceae) is shrub
belonging to family Melastomataceae and widely
distributed at Bidoup- Nui Ba National Park, Lam Dong
province, Vietnam. The ethnobotany information revealed
that local people use its leaves and young shoot to treat
diarrhea. Since there is no report on M. septentrionalis as
an anti-diarrheal herb, the present study is conducted to
evaluate its bioactivity, toxicity and anti-diarrheal activity.
MATERIALS AND METHODS-
Plant identification-
Medinilla septentrionalis (W.W. Sm) H.L. Li obtained from
BiDoup– Nui Ba National Park, Lam Dong Province,
Vietnam was identified by the Department of Ecology and
Evolutionary Biology, Faculty of Biology- Biotechnology,
University of Science, Vietnam.
Plant identification was carried out by comparison of the
Morphology of reproductive (flowers, fruits) and vegetative
organs (leaves, stems, rhizomes) of specimens with those
described in taxonomy references and with those in digital
herbaria: Missouri Botanic Garden, Royal Botanic Gardens
-Kew, Berlin-Dahlem Botanical Garden. The voucher
specimen was deposited at the Herbarium of University of
Science, Ho Chi Minh City Vietnam National University.
Preparation of M. septentrionalis ethanolic extract
(MsEE)-
Medinilla septentrionalis was collected from BiDoup – Nui
Ba National Park, Lam Dong Province, Vietnam. Young
shoots and leaves were dried under sunlight and powdered
and prepared as modified procedure of Milosevic et al. [6].
The sample was extracted with ethanol 70% by immersion
method at room temperature for 48 hours. The extract was
filtered and evaporated at 400C, using rotary evaporator to
constant weight. Yield of the extract was 21.41% (w/w).
The extract was then dissolved in 1% dimethyl sulfoxide
(DMSO) and stored in sterilized bottle in 40C.
Preparation of indicator bacteria and animals-
Two groups of indicator bacteria that included 19 strains of
pathogenic bacteria (4 strains of Salmonella spp.; 3 strains
of Shigella spp.; 4 strains of Escherichia coli; 3 strains of
Vibrio spp. and 5 strains of other pathogenic bacteria) and 7
strains of useful bacteria (4 strains of Lactobacillus spp.
and 3 strains of Bacillus spp.) were used to evaluate antibacterial
activity of M. septentrionalis ethanolic extract.
Albino mice (25-30 g) were used to evaluate anti-diarrheal
activity of M. septentrionalis ethanolic extract (MsEE). All
the animals were housed in glass cages in laboratory
conditions at least 1 week before performing experiment.
Antibacterial testing using well diffusion agar
method-
The M. septentrionalis ethanolic extract was evaluated
antibacterial activity by well diffusion agar method [7].
Indicator bacteria were enriched at temperature 370C for 24
hours. 100µl of diluted bacteria (106 cfu ml–1) solution was
spread on agar medium until drying. Then, wells (6 mm
diameter) were made in each of plate by using sterile metal
cylinders. 100µl of the M. septentrionalis ethanolic extract
(100 mg ml–1) were added into the wells. Control
experiment comprised inoculums with 1% DMSO. Plates
were incubated at 37.0 ± 0.10C for 24 hours. The diameter
of the inhibition zone (mm) was measured. Each
experiment was triplicated and collected data were subjected
to statistical analysis.
Acute toxicity study-
The acute toxicity of M. septentrionalis ethanolic extract
was determined in mice. Mice were fasted for 18 hours and
randomly divided into five groups (6 mice per group).
Different doses of plant extract (2500, 5000, 7500 and
10000 mg kg–1) were separately oral administered to the
mice. The fifth groups of animals as control was
administered DMSO 1% (2 ml kg–1 body). All of animals
were observed over a period of 5 days for deaths and signs
of acute toxicity.
Castor oil-induced diarrhea in mice-
Mice were fasted for 10-12 hours then divided into six
groups (6 mice per group). Group of control animals was
administered DMSO 1% (2 ml kg–1 body). The second
group received standard drug, loperamide (3 mg kg–1 body)
orally as a suspension. The plant extract was administered
orally at the doses 63, 125 250, 500, 1000 mg kg–1 body to
the five remain groups, separately. After 30 min of
treatment, the animals of each group were received 0.4 ml
castor oil orally. Then, they were housed separately in cage
over clean filter paper. Diarrhea episodes were observed for
a period of 4 hours.
During this period, the first defecation time, the defecation
animal number at each group and the cumulative wet fecal
mass was recorded. The percent of diarrheal inhibition (PI)
was defined as formula:
The results of treated groups were compared with control group to evaluate diarrheal treatment effectiveness of M. septentrionalis ethanolic extract.
Castor oil-induced enteropooling test- Mice were treated with castor oil and plant extract as mentioned above. After 1 hour of castor oil administration, all animals were sacrificed by overdose of diethyl ether. The small intestine (from the pylorus to the caecum) was dissected out and weighed. Its content was collected into cylinder and volume measured. The empty intestine was weighed again then percentage reduction of intestine secretion (volume) was calculated.
Small intestine transit test in mice- Mice were fasted and treated with plant extract as mentioned above. 30 minutes after plant extract administrated, the mice were administered 0.4 ml castor oil, followed by 0.2 ml charcoal meal (3% charcoal suspension in carboxymethyl cellulose (CMC) 0.5% (w/v). Then, each animal was housed separately in cage. After 1 hour, all animals were sacrificed by overdose of diethyl ether then the small intestine (from the pylorus to the caecum) was dissected. Charcoal meal moving distance was measured and then expressed as a percentage of the distance from the pylorus to the caecum.
Preliminary phytochemical analysis- The Medinilla septentrionalis ethanolic extract was chemically tested for the presence of different constituents including carbohydrates, alkaloids, saponins, cardiac glycosides, anthraquinone glycosides, flavonoids, phenolic compounds, tannin, steroids and amino acids by using standard methods [8].
STATISTICAL ANALYSIS- Values were expressed as mean ± standard deviation. Mean values were evaluated by Analysis of Variance. Duncan test was used to determine the statistical significance (P < 0.05).
RESULTS AND DISCUSSION
Identification of M. septentrionalis- M. septentrionalis is shrub of 1–5(–7) meter tall with many brown branches erect or scrambling. Leaves have petiole of 0.4–1 mm long; leaf blade is lanceolate or ovate-lanceolate in shape, 7-10 × 2–3 cm, papery; its apex is long-acuminate, its base is obtuse to subrounded and its margin is sparsely serrulate just around in the apex area; five veins are found from the bottom, in which the secondary veins (2) are on each side of mid-vein. From 3 to five flowers, 2.5-5 cm, are found in small terminal cymose panicles and in lateral cymes. The receptacle is in hypanthium cup-shaped, 4–4.5 mm, sparsely ciliate papules. Calyx lobes have four, in green color and inconspicuous. Petals have four, in light pink or purplish red, triangular-ovate, 8–10 mm. Their stamens are eight, equal or nearly equal: four longer (outer) stamens and four shorter (inner) stamens; the connectives are slightly elongated. The ovary is ovoid. Fruit is berry, globose-ovoid, 6-7 × 4-5 mm (Fig. 1). It blooms during June to September and produces fruit from February to May.
The plant is found in dense forests, forest margins and damp shady areas of Vietnam, China, Myanmar and Northern Thailand.
Fig. 1 Medinilla septentrionalis plant
Anti-diarrheal activity of M. septentrionalis- In this study, defecation in castor oil-induced diarrhea mice, which were treated by MsEE, was significantly reduced. While loperamide (3 mg kg–1) inhibited 80.85% of mice defecation, the crude extract of M. septentrionalis inhibited 41.49% of mice defecation at 63 mg kg–1 used concentration. The MsEE defecation inhibitory activity was dose-dependence when it was clearly increasing in high MsEE dose of treatment (Table 2). In castor oil-induced diarrheal mouse model, anti-diarrheal activity may be attributed to an anti-electrolyte permeability action and intestine transit. In our experiment, castor oil-induced enteropooling was observed in all experimental mice and it was strongly weakened by MsEE treatment at 63 mg kg–1 dose. The MsEE had shown a dose–dependent effect in reduction in intestinal weight and volume (Table 3).
Besides, MsEE also strongly reduced the charcoal meal transit rate in castor oil-induced mice (Table 4).
The result was shown that the MsEE had the high anti-diarrheal activity. The cause of diarrhea is characterized by excessive secretion water and electrolytes into intestine lumen, exudation of protein and fluid from the mucosa and altered intestinal motility, resulting in rapid transit time and an increase wet feces. Castor oil stimulates secretion of fluid and electrolytes and increases the intestine transit [9]. These results showed that MsEE was clearly effective to treat diarrhea in castor oil-induced mice model by reducing wet feces, inhibiting secretion fluid and gastrointestinal propulsion. Compared to other studies, the anti-diarrheal potential of M. septentrionalis is roughly equivalent to other herbs such as Moringa oleifera [1, 5], Vinca major [10], Alangium salviifolium [11], Lepidium sativum [12], Dillenica indica [13].
Table 1 Antibacterial activity of M. septentrionalis ethanolic extract
| Indicator bacteria | Diameter of inhibition zone (mm) | Indicator bacteria | Diameter of inhibition zone (mm) |
|---|---|---|---|
| S. dublin | 14.33 ± 0.76 | E. coli O157:H7 | 13.33 ± 0.76 |
| S. enteritidis | 13.33 ± 1.04 | E. coli 0208 | 13.83 ± 0.29 |
| S. typhii | 14.50 ± 0.00 | E. Coli | 13.33 ± 0.76 |
| S. typhimurium | 13.83 ± 1.04 | ETEC | 14.67 ± 0.58 |
| S. boydii | 14.67 ± 0.58 | V. alginolyticus | 14.00 ± 1.00 |
| S. flexneri | 14.50 ± 0.58 | V. cholerae | 12.50 ± 0.87 |
| S. sonnei | 13.67 ± 0.29 | V. parahaemolyticus | 12.17 ± 0.29 |
| L. innocua | 14.17 ± 0.29 | S. aureus | 14.83 ± 0.28 |
| L. monocytogenes | 13.83 ± 0.76 | E. feacalis | 13.83 ± 0.76 |
| P. aeruginosa | 13.83 ± 0.29 | ||
| B. subtilis | NA | L. lactis LB1 | |
| B. subtilisBS1 | NA | L. plantarum LB2 | NA |
| B. licheniformisBS2 | NA | L. plantarumSC01 | NA |
Table 2 Defecation inhibitory activity of M. septentrionalis ethanolic extract
| Treatment | Onset of diarrhea (min) | Animals with diarrhea | No. of faeces in 4h (g) | % inhibition of defecation |
|---|---|---|---|---|
| Castor oil + DMSO 1% (2 ml kg–1) | 85.33 ± 11.54a | 6/6 | 0.78 ± 0.12a | |
| Castor oil + Loperamide (3 mg kg–1) | 220.50 ± 7.78d | 2/6 | 0.15 ± 0.07e | 80.85 |
| Castor oil + MsEE (1000 mg kg–1) | 206.50 ± 6.36cd | 2/6 | 0.23 ± 0.03de | 71.28 |
| Castor oil + MsEE (500 mg kg–1) | 182.00 ± 10.15c | 3/6 | 0.27 ± 0.06cde | 65.96 |
| Castor oil + MsEE (250 mg kg–1) | 146.50 ± 9.15b | 4/6 | 0.35 ± 0.06bcd | 55.32 |
| Castor oil + MsEE (125 mg kg–1) | 144.80 ± 4.87b | 5/6 | 0.43 ± 0.16bc | 45.11 |
| Castor oil + MsEE (63 mg kg–1) | 136.00 ± 11.26b | 6/6 | 0.46 ± 0.11b | 41.49 |
Table 3 Enteropooling reduction in M. septentrionalis ethanolic extract treated mice
| Treatment | Weight intestinal content (g) | % inhibition in weight of intestinal content |
|---|---|---|
| Castor oil + DMSO 1% (2 ml kg–1) | 1.12 ± 0.18a | - |
| Castor oil + Loperamide (3 mg kg–1) | 0.25 ± 0.09c | 76.87 |
| Castor oil + MsEE (1000 mg kg–1) | 0.35 ± 0.19bc | 68.66 |
| Castor oil + MsEE (500 mg kg–1) | 0.42 ± 0.21bc | 62.69 |
| Castor oil + MsEE (250 mg kg–1) | 0.45 ± 0.08bc | 59.70 |
| Castor oil + MsEE (125 mg kg–1) | 0.50 ± 0.37bc | 55.22 |
| Castor oil + MsEE (63 mg kg–1) | 0.70 ± 0.18b | 40.30 |
Table 4 Effect of M. septentrionalis ethanolic extract on small intestine transit in mice
| Treatment | Mean intestine length (cm) | Mean distance travel by charcoal (cm) | % Movement | % Inhibition |
|---|---|---|---|---|
| Castor oil + DMSO 1% (2 ml kg–1) | 43.58 ± 5.04 | 35.08 ± 1.39 | 81.28 ± 8.63a | 18.72 ± 8.63 |
| Castor oil + Loperamide (3 mg kg–1) | 39.25 ± 6.21 | 13.50 ± 2.12 | 34.91 ± 4.99de | 65.09 ± 4.99 |
| Castor oil + MsEE (1000 mg kg–1) | 51.00 ± 8.83 | 17.92 ± 12.14 | 35.39 ± 6.72d | 64.61 ± 6.72 |
| Castor oil + MsEE (500 mg kg–1) | 47.25 ± 4.98 | 20.67 ± 4.87 | 43.47 ± 9.81cd | 56.53 ± 9.81 |
| Castor oil + MsEE (250 mg kg–1) | 47.17 ± 8.29 | 25.08 ± 6.97 | 52.63 ± 4.95bcd | 47.37 ± 4.95 |
| Castor oil + MsEE (125 mg kg–1) | 42.25 ± 4.27 | 24.17 ± 2.48 | 57.96 ± 11.27bc | 42.04 ± 11.27 |
| Castor oil + MsEE (63 mg kg–1) | 40.00 ± 6.19 | 28.58 ± 5.77 | 71.40 ± 11.31ab | 28.60 ±11.31 |
Acute animal study- In acute toxicity study, mice were treated by MsEE at several different high doses (2500, 5000, 7500 and 10000 mg kg–1). Our observation on all treated mice demonstrated that during 5 days after MsEE oral administration no significant toxicity signs and mortality were obtained. The treated mice were holding normal physiology condition, body temperature.
Preliminary phytochemical analysis- The preliminary phytochemical screening of M. septentrionalis ethanolic extract showed the presence of carbohydrates, saponins, cardiac glycosides, flavonoids, phenolic compounds, tannins and steroids (Table 5).
The data on indicator bacteria and castor oil-induced mouse model strongly contributed evidence of M. septentrionalis antibacterial and anti-diarrheal activity. Coincidently, the preliminary phytochemical screening data provided a sight of mechanism on study of M. septentrionalis activity as an anti-diarrheal herb. It is well known that flavonoid; phenolic compound and tanin are antibacterial compounds [14-15]. Our data also demonstrated that M. septentrionalis ethanolic extract contains those mentioned compounds. Taken together, it suggested that antibacterial activity of the plant might due to the flavonoid, tannin and phenolic compounds. Furthermore, both tannin and flavonoid can precipitate protein of the electrolyte and reduce small intestine transit and intestinal secretion [16-17], the compounds may play function in M. septentrionalis anti-diarrheal activity.
Table 5 Phytochemical screening of M. septentrionalis ethanolic extract
| Chemical test | Results | Chemical test | Results |
|---|---|---|---|
| Test for carbohydrate Molisch’s test Fehling’s test Barfoed’s test |
+ + + |
Test for amino acid Ninhydrin test |
- |
| Test for flavonoid Alkaline reagent test Shinoda’s test Ferric chloride test |
+ + + |
Test for phenolic compound Lead acetate test Gelatin test |
+ + |
| Test for tannin Ferric chloride test Lead acetate test |
+ + |
Test for steroids Salkowski’s test Libermann Burchard test |
+ + |
| Test for alkaloid Mayer’s test Dragendorff’s test Hager’s test Wagner’s test |
- - - - |
Test for anthraquinone glycosides Borntrager’s test |
- |
| Test for saponin Foam test |
+ |
Test for cardiac glycosides Legal’s test Keller Killiani’s test |
+ + |
CONCLUSION- M. septentrionalis are used to treat diarrhea at ethnic minorities in Vietnam. In our study, diarrhea- related bacteria were strongly inhibited by M. septentrionalis ethanolic extract. The data of assay on castor oil-induced mouse model strongly contributed evidence of M. septentrionalis anti-diarrheal activity. Coincidently, the preliminary phytochemical screening data provided a sight of mechanism on study of M. septentrionalis activity as an anti-diarrheal herb. Since M. septentrionalis has not been reported as anti-diarrheal herb yet, the present study strongly demonstrated that M. septentrionalis is effective in the treatment of diarrhea either by its antibacterial or antidiarrheal activity.
ACKNOWLEDGMENT- This research is funded by Vietnam National University Ho Chi Minh City (VNU-HCM) under grant number B2014-18-04 and sampling supported by the BiDoup– Nui Ba National Park, Vietnam. We specially thanked to Dr Ho Viet Anh, University of Science, VNU-HCMC, for great support on techniques and experiments.
REFERENCES
- Saralaya MG, Patel P, Patel M, Roy SP, Patel AN. Antidiarrheal activity of Methanolic extracts of Moringa oleifera Lam Roots in Experimental animals. Int J Pharm Res, 2010; 2(2): 35–39.
- Guandalini S and Vaziri H. Diarrhea: Diagnostic and Therapeutic Advances. London: Springer: 2011. p. 1-31.
- Sarin RV and Bafna PA. Herbal Antidiarrheals: A review. Int J Res Pharm Biomed Sci, 2012; 3(2): 637-649.
- Shahid AA. Biological activities of extracts and isolated compound from Bauhinia galpinii (Fabaceae) and Combretum vendae (Combretaceae) as potential antidiarrhoeal agents. University of Pretoria, South Africa, 1998.
- Lakshminarayana M, Shivkumar H, Rimaben P, Bhargava VK. Antidiarrhoeal activity of leaf extract of Moringa oleifera in experimentally induced diarrhoea in rats. Int J Phytomedicine, 2011; 3: 68–74.
- Milosevic T, Solujic S, Sukdolak S. In vitro study of ethanolic extract of Hypericum perforatum L. on growth and sporulation of some bacteria and fungi. Turk J Biol, 2007; 31: 237–241.
- Sen A, Batra A. Evaluation of antibacterial activity of different solvent extracts of medical plants: Melia azedarach L. Int J Curr Pharm Res, 2012; 4(2): 67–73.
- Jayaveera KN, Yoganandham RK, Govindarajula Y, Kumanan R. Phytochemical screenings, antibacterial activity and physical chemical constants of ethanolic extract of Euphorbia thymifolia Linn. Int J Pharm Pharm Sci, 2010; 2(3): 81–82.
- Das AK, Rohini RM, Hema A. Evaluation of anti-diarrhea activity of Rhizophora mucronata bark extracts. J Alter Med, 2008; 7(1): 1-6.
- Rajput MS, Nair V, Chauhan A, Jawanjal H, Dange V. Evaluation of Antidiarrheal Activity of Aerial Parts of Vinca major in Experimental Animals. Middle East J Sci Res, 2011; 7(5): 784–748.
- Zahan R, Mosaddik MA, Barman RK, Wahed MII, Haque ME. Antibacterial and antidiarrhoeal activity of Alangium salviifolium Wang flowers. Mol Clin Pharmacol, 2012; 2(1):34–43.
- Manohar D, Lakshman K, Shylaja H, Viswanatha GL, Rajesh S, Nadakumar K. Antidiarrheal activity of methanolic extracts of seeds of Lepidium savatum. J Nat Remedies, 2009; 9(2): 197–201.
- Islam MM, Pia RS, Sifath-E-Jahan K, Chowdhury J, Akter F, Parvin N, Akter S. Antidiarrheal activity of Dillenia indica bark extract. Int J. Pharm Sci Res, 2013; 4(2): 682-688.
- Silva NNC, Fernandes Junior A. Biological properties of medicinal plants: a review of their antimicrobial activity. JVATiTD, 2010; 16(3): 402-413.
- Gobalakrishnan R, Kulandaivelu M, Bhuvaneswari R, Kandavel D, Kannan L. Screening of wild plant species for antibacterial activity and phytochemical analysis of Tragia involucrata L. J Pharm Analysis, 2013; 3(6): 460-465.
- Rajeswari K, Kumar AR, Rathinam KMS. Phytochemical and anti diarrhoeal activity of Hippocratea Africana roots. Indian J Res Pharm Biotech, 2014; 2(4): 1357-1359
- Sai BN, Chand CVG, Krishna GR, Kumar AR, Reddy AJR, Vallabh V. Phytochemical and anti diarrhoeal activity of extract of Morinda citrifolia. Int J Pharm Chem Biol Sci, 2015; 5(1): 138-140.
| International Journal of Life-Sciences Scientific Research (IJLSSR) Open Access Policy Authors/Contributors are responsible for originality, contents, correct references, and ethical issues. IJLSSR publishes all articles under Creative Commons Attribution- Non-Commercial 4.0 International License (CC BY-NC). https://creativecommons.org/licenses/by-nc/4.0/legalcode |
| How to cite this article: Nhut PM, Minh Ai NX, Phuong Thao DT: Anti-Diarrheal Evaluation of Medinilla septentrionalis. Int. J. Life. Sci. Scienti. Res., 2017; 3(1): 832-837. DOI:10.21276/ijlssr.2017.3.1.14 Source of Financial Support: VNU-HCM, Vietnam, Conflict of interest: Nil |