In-Vitro Study of Antimicrobial and Antiproliferative activity of Cyanogenic glycoside extracted from Bamboo shoot of Bambusa arundinacea

 

Binku Mondal^1,2

¹Department of Home Science, Jawaharlal Nehru Rajkeeya Mahavidyalaya, Port Blair,

South Andaman – 744104, India.

²Department of Food Science and Nutrition, Periyar University, Salem, Tamilnadu, India – 636011.

 

ABSTRACT:

Bambusa arundinacea is a common bamboo species found in Andaman and Nicobar islands.  There are over 1200 species of bamboo belonging to 75 genera which has been documented worldwide. Bamboo plants especially the leaves and shoots are known to have cyanogenic glycosides like taxiphyllin and amygdalin apart from vitamin C and other components. Immature bamboo shoots are consumed as vegetable in several asian countries. In the present study the crude extract of Bambusa arundinacea in various solvents was performed. Further preliminary phytochemical screening of the extract was done. Based the results of phytochemical screening, methanol extract was selected for further study. HPLC analysis was done confirm the presence of Amygdalin. GC-MS Analysis of methanol extract of B.arundinacea shows the presence of 40 compounds. The major compounds found in the methanol extracts based on the retension time were beta.-Sitosterol; 2-Methyl-7-phenylindole, 1-(Methylamino)anthraquinone, (R)-(-)-14-Methyl-8-hexadecyn-1-ol and 2-Ethylbutyric acid, nonadecyl ester. Antimicrobial activity of the extracts were tested using two Gram positive bacteria namely  S. aureus and pneumonia and two Gram negative bacteria viz. Escherichia coli and Streptococcus mutant. The zone of inhibition was maximum in acetone and ethylacetate extracts while it was minimum for methanol extract. This might be due to the presence of taxiphyllin in the acetone and ethylacetate extract which are relatively non-polar as compared to methanol. To determine whether the inhibition of cell proliferation by methanolic extract from bamboo shoot was due to the induction of apoptosis, PC3 cell exposed to methanol extract were stained with acridine orange/ethidium bromide. The results show that at dose of 150 μg/mL, methanolic extract showed increased percentages of early apoptotic cells. Based on the results of the study, methanol extract of Bambusa arundinacea is a rich source of secondary metabolites, with presence of high levels of flavanoids and tannins. Presence of amygdalin in methanol extract is confirmed by HPLC analysis. Though the extract has a comparatively low antimicrobial activity yet the high anti-apoptotic activity of the extract coupled with a rich antioxidant ativity makes it an ideal candidate as a source of drug for cancer therapy.

 

 

INTRODUCTION:

Plants are important source of drugs, for the benefit of human health. Bioactive molecules and their combinations have been traditionally used to treat chronic and infectious diseases^1-2. Plants are a rich source of secondary metabolites and many of these have several bioactive properties, so plant based drugs are continuously in great demand. According to a report by WHO for primary health care needs more than 80% of the world’s population depends on traditional medicine³. In order to use plants to cure various ailments through traditional medicine it is essential to evaluate the use of plants in a systematic manner for biological activity screening of medicinal plants are in its utmost need. Bamboo is a forest tree grass belonging to family poaceae and subfamily  Bambuseae. Over 1250 species under 75 genera of bamboos species are spread all over the world⁴. In India, there are about 136 species under 23 genera⁵. In China, Japan and other East Asian countries, bamboo shoots are used as a major food source⁶. According to ancient Chinese medicinal books such as “Ben Jing Feng Yuan,” “Yao Pin Hua Yi,” and Jing Yue,” bamboo shoots are proclaimed to have beneficial effect on human health. The shoots are thought to help in digestion, promotes peristaltic movement and is  helpful in treating constipation. Freshly harvested juvenile bamboo shoots have shown high amount of amino acids, proteins, carbohydrates, vitamins and minerals and a low content of fat. The vitamin and mineral content starts decreasing when the bamboo shoots starts aging meanwhile the dietary fiber and moisture content starts increasing⁷. The most commonly used edible bamboo species are Bambusa pallida, Bambusa tulda, Bambusa polymorpha, Bambusa balcooa, Dendrocalamus hamiltonii, Dendrocalamus giganteus and Melocanna bambusoides⁵. Bamboo shoots have high potassium content which helps to maintain normal blood pressure. The shoots are also known to reduce body fat and helps prevent cardio vascular diseases (CVDs). Recent studies have shown that bamboo extracts can effectively reduce the free radicals and has anti-inflammatory property^8-9. In China, Japan and other East Asian countries, bamboo shoots are used as a major food source⁶. Pyrolysates derived from bamboo promisely have antimicrobial and antifungal activities¹⁰ which protect the neurons from oxidative stress. To treat ischemic injury and antiapoptotic effects pyrolysates can be used as supplement derived from 3 bamboo species viz. Phyllostachys bambusoides, P. nigra and P. pubescens respectively¹¹. Bamboo shoots are used in traditional Chinese medicine which induces the uterine contractions and helps to ease the labor and expulsion of the placenta. To treat respiratory diseases bamboo shoot decoction is used along with honey.

 

MATERIALS AND METHODS:

Collection of plant material:

The shoots of Bambusa arundinacea of Bambusaceae family were collected from the forests of Little Andaman which is one of the Island of Andaman and Nicobar Islands. The shoot samples were cut and washed with potable water and then kept in room temperature for draining the water. It is then packed in polythene bag and transported.

 

Preparation of bamboo shoot powder:

The bamboo shoots were washed in tap water, then cut into pieces, boiled for 30 minutes remove the hydrocyanic acid from the sample¹². The water was drained and the samples were washed 4 times with distilled water. After washing the samples were air dried. The dried sample was powdered in electric blender, packed and stored for further analysis.

 

Extraction of active compounds:

For extraction of secondary metabolites several solvents like water, ethanol, methanol, ethyl acetate, acetone, and hexane based on polarity of solvents. Soxhlet apparatus was used for the extraction process¹³. The extracts were vaccum dried and stored at 4°C in a refrigerator for further studies.

 

Phytochemical screening:

Phytochemical screening of was done to identify the bioactive components present in the extracts as per method¹⁴. 

 

Antimicrobial activity:

The agar well diffusion method was used to test the antimicrobial activity of the extracts. Staphylococcus aureus, Echerichia coli, Streptococcus mutant and Pseudomonas were used in the study. These were maintained in nutrient agar media for 24 hours. The nutrient agar media was prepared and poured in sterilized petriplates. After that the microorganisms were spread over the plates and100 μL of all the extracts were loaded in the well on the agar media. The plates were kept in an incubator for 24 hours at 37˚C. The zone of inhibition was measured in mm as determined by¹⁵.

 

Anti-apoptotic property of methanol extract on prostate cancer (PC3) cell line:

Cell culture and reagents : Prostatic cell line PC3 were maintained in RPMI 1640 supplemented with 10% heat-inactivated fetal calf serum antibiotics, and 2 mM L-glutamine, Phenylacetic acid (Sigma Chemical Co., St. Louis, MO) was dissolved in distilled water, brought to pH 7.0 by the addition of NaOH, and stored in aliquots at -20°C till used.

 

Determination of Apoptosis by AO/EB Staining:

DNA-binding dyes AO and EB (Sigma, USA) were used for the morphological detection of apoptotic and necrotic cells. AO is taken up by both viable and non-viable cells and emits green fluorescence if intercalated into double stranded nucleic acid (DNA). EB is taken up only by non-viable cells and emits red fluorescence by intercalation into DNA. The cells were cultured in Dulbecco's modified Eagles medium and grown to 60-70% confluency and treated with liver extracts at a final concentration of 100mcg/ml for 24 h, the cells were washed with cold PBS and then stained with a mixture of AO (100μg/ml) and EB (100μg/ml) at room temperature for 10 minutes. The stained cells were washed twice with 1X PBS and observed by a fluorescence microscope in blue filter of fluorescent microscope.

 

RESULT:

Yield of extracts of Bambusa arundinacea:

Compared to all the extracts methanolic extract had high yield of 31.43 % followed by ethanolic extract with 27.34 %, and least being acetone extract with 14.69 %

 

Qualitative analysis of bamboo shoot powder:

The phytochemical screening of bamboo shoots powder was carried out in solvent extracts namely, aqueous, hexane, ethanol, methanol, ethyl  acetate and acetone. Phytoconstituents such as steroids, terpenoids, fatty acid, tannins, saponins, anthocyanins, leucoanthocyanins, coumarins and emodins were analysed. The results show that methanol extract showed the presence of most compounds while hexane extract showed the presence of only steroids (Table1).

 

GC-MS analysis of Bamboo shoot methanolic extract:

Totally 40 compounds were identified in Bambusa arundinacea shoot. The active principles with their retention time (RT), molecular weight (MW) and peak area (%) are given in (table 2). The major compounds found in the methanol extracts based on the retension time were beta.-Sitosterol; 2-Methyl-7-phenylindole, 1-(Methylamino)anthraquinone, (R)-(-)-14-Methyl-8-hexadecyn-1-ol and 2-Ethylbutyric acid, nonadecyl ester. In total about forty compounds were identified in the GC-MS analysis.  These molecules are known to anti-lipedemic as well as anti cancer activities.

 

Antimicrobial activity:

The antimicrobial activities of all the different extracts. The level of inhibition was observed with two Gram positive bacterial species  S. aureus and pneumonia and two Gram negative bacterial species Escherichia coli and Streptococcus mutant. The fresh extract of Bambusa arundinacea was used in different concentration which showed maximum zone of inhibition. The maximum activity was observed against S. aureus, gram positive bacteria were shown in (Table. 3).  The highest activity was observed in acetone extract and ethyl acetate extract of 25mm in 100μl.  In methanolic extract the zone of inhibition was observed of 17mm in 100μl. In Ethanolic extract it is 15mm was observed in 100μl. In aqueous extract the maximum zone was observed in 100μl. However, very less activity was observed in hexane extract against S. aureus.

 

However in Pseudomonas bacteria the maximum zone of inhibition in acetone extract in 100μl of 30mm. In ethyl acetate the zone of inhibition is 27mm was observed in 100μl respectively were as in ethanolic extract the zone of inhibition is 9mm in 100μl and in methanolic extract it is 2mm in 100μl. Finally in hexane and aqueous extract there is no zone formation.

 

In E. coli bacteria acetone extract has the highest zone of inhibition of 25mm in 100μl, whereas in ethyl acetate extract the zone of inhibition is 20mm in 100μl. In methanolic extract the zone of inhibition is 17mm in 100μl. And in ethanolic extract it is 4mm in 100μl. in hexane and aqueous extract there is no zone formation it has been observed.

 

In S.mutant bacteria both ethyl acetate extract and acetone extract has the same zone of inhibition of 25 mm in 100μl. Whereas in aqueous extract in 100μl concentration the zone of inhibition is 1mm. in methanolic and ethanolic extract the zone of inhibition is not observed.  The shoots of bamboo inhibit the growth of Microorganism. The gram positive bacteria (S.aureus) were more sensitive than gram negative bacteria (K.pneumonia). Significant inhibition was recorded with methanol and aqueous extracts. It is important to note that the methanol extracts can inhibit the growth in S.aureus which is a biogenic amine producer and is a major cause of cutaneous infections, furunculosis, impetigo, arthritis, and toxinoses, such as food poisoning, septic shock, scalded skin syndrome and toxic shock syndrome¹⁶.

 

Table 1: Preliminary qualitative analysis or Phytochemical screening of Bamboo shoots powder

Phytochemical components	Aqueous Extract	Hexane Extract	Ethanol Extract	Methanol Extract	Ethyl Acetate Extract	Acetone Extract
Steroids	+	+	+	+	+	+
Terpenoids	+	-	+	+	+	-
Fatty acid	-	-	-	-	-	-
Tannins	+	-	+	+	+	-
Saponins	+	-	+	+	-	+
Anthocyanins	-	-	+	+	+	+
Leucoanthocyanins	-	-	+	+	-	+
Coumarins	+	-	-	+	+	-
Emodins	-	-	+	+	-	+

Legends: + = positive, - = negative

 

Table 2: GC-MS analysis of Methanol extract of Bambusa arundinacea

S. No	Peak No.	Area %	Retention Time	Compound name
1	1	0.29	10.094	Butanedioic acid, monomethyl ester,
2	2	4.98	10.892	4H-Pyran-4-one, 2,3-dihydro-3,5- dihydroxy-6-methyl-
3	3	0.56	11.976	Benzofuran, 2,3-dihydro-
4	4	0.38	12.419	1,2,3-Propanetriol, 1-acetate
5	5	0.43	13.130	Phenol, 4-(methoxymethyl)-
6	6	0.34	13.503	Acetamide, N,N-diethyl
7	7	0.71	13.888	Benzenemethanol, 3-hydroxy
8	8	32.06	14.150	Cyanogl
9	9	0.39	14.244	Benzaldehyde, 4-hydroxy-
10	10	2.30	14.302	DL-Proline, 5-oxo-, methyl ester
11	11	1.41	14.937	Propanamide, N,N-dimethyl-
12	12	0.94	15.281	Methylparaben
13	13	1.15	15.578	D-mandelonitrile-β-gentiobioside
14	14	0.60	15.631	Propanoic acid, 3-hydroxy-3-phenyl
15	15	0.48	16.068	Benzoic acid, 4-hydroxy-
16	16	0.54	16.558	4-(2-Methoxyethyl)phenol
17	17	0.34	17.054	Ethyl N-(o-anisyl)formimidate
18	18	5.41	17.531	Butyraldehyde, semicarbazone
19	19	0.34	17.572	Silver butanoate
20	20	9.91	17.636	Butanoic acid, Pentyl ester
21	21	0.39	18.732	Tetradecanoic acid
22	22	0.45	18.808	2-Propenoic acid, 3-(4-hydroxyphenyl)-, methyl ester
23	23	2.78	20.480	Hexadecanoic acid, methyl ester
24	24	4.63	20.853	n-Hexadecanoic acid
25	25	0.43	21.366	Pyridine, 2-ethyl-4,6-dimethyl-
26	26	4.65	22.147	9,12-Octadecadienoic acid (Z,Z)-, methyl ester
27	27	1.35	22.206	7, 10, 13-Hexadecatrienoic acid, methyl ester
28	28	3.69	22.520	9,12-Octadecadienoic acid (Z,Z)-
29	29	3.66	22.567	9,12-Octadecadienoic acid (Z,Z)-
30	30	2.74	22.759	Octadecanoic acid
31	31	0.33	23.843	O-Ethyl S-2-dimethylaminoethyl ethylphosphonothiolate
32	32	0.32	24.147	1,1́-Bicyclohexyl-2,2́-diol
33	33	0.64	25.289	S-[2-[N,N-Dimethylamino]ethyl]morpholine-N-cabonylthiocarbohydroximate
34	34	1.84	25.732	1H-1,2,4- Triazole-1-acetamide,N-3-methoxyphenyl)-3-nitro-
35	35	1.54	25.796	Hexadecanoic acid, 2-hydroxy-1-(hydroxymethyl) ethyl ester
36	36	2.12	27.090	2-Ethylbutyric acid, nonadecyl ester
37	37	3.32	27.183	(R)-(-)-14-Methyl-8-hexadecyn-1-ol
38	38	0.33	27.533	1-(Methylamino)anthraquinone
39	39	0.29	28.401	2-Methyl-7-phenylindole
40	40	0.95	28.699	.beta.-Sitosterol

 

Table 3: Antibacterial activity of  Bambusa arundinacea shoot solvent extract.  The zone of inhibition is given in Diameter(mm)

Extract/ Concentration	Test Organism	25 μl	50 μl	75 μl	100μl
Methanol	Staphylococcus aureus	9	10	13	17
	Pseudomonas aeruginosa	No zone	No zone	1	17
	Escherichia coli	No zone	No zone	No zone	17
	S.mutant	No zone	No zone	No zone	No zone
Ethanol	Staphylococcus aureus	4	9	10	15
	Pseudomonas aeruginosa	2	7	8	9
	Escherichia coli	No zone	No zone	1	4
	S.mutant	No zone	No zone	No zone	No zone
Ethyl Acetate	Staphylococcus aureus	5	7	20	25
	Pseudomonas aeruginosa	12	13	25	27
	Escherichia coli	13	15	17	2
	S.mutant	12	15	17	25
Acetone	Staphylococcus aureus	13	14	20	25
	Pseudomonas aeruginosa	11	13	17	30
	Escherichia coli	12	15	2	25
	S.mutant	12	15	2	25
Hexane	Staphylococcus aureus	3	5	6	9
	Pseudomonas aeruginosa	No zone	No zone	No zone	No zone
	Escherichia coli	No zone	No zone	No zone	No zone
	S.mutant	No zone	No zone	No zone	No zone
Aqueous	Staphylococcus aureus	3	5	9	13
	Pseudomonas aeruginosa	No zone	No zone	No zone	No zone
	Escherichia coli	No zone	No zone	No zone	No zone
	S.mutant	No zone	No zone	No zone	No zone

 

Apoptosis AO/EB double staining:

AO\EB dual stain was employed to identify the apoptotic and necrotic cells. The Prostate cancer cell line P3 cells treated with extracts showed more apoptotic cells than control group. To determine whether the inhibition of cell proliferation by methanolic extract from bamboo shoot was due to the induction of apoptosis, we assessed the latter with the acridine orange/ethidium bromide method. Prostate cancer cell line P3 cell treated with 150 μg/mL methanolic extracts of bamboo shoot showed increased percentage of early apoptotic cells (Figure 1 and Table 4).

 

Figure 1: Determination of apoptosis

 

Table 4: MTT assay of Bamboo shoot extract

 

DISCUSSION:

Plant are rich source of secondary metabolites. These secondary metabolites have a variety of applications as drug in human health and in development of ecofriendly insecticides^17-18. Flavanoids, terpenes, phenolics, essential oil and fats are important components found in secondary metabolites. Among plants bamboo is important as it is a source of antioxidant and is consumed as food in several Asian countires^19-20-21. Bamboo is a type of forest tree grass belonging to family Poaceae. There are over 1250 species under 75 genera of bamboos are spread all over the world⁴. Around 2.5 billion people are directly involved in the production and consumption of bamboo. In India, there are about 136 species under 23 genera¹⁹.

 

Bamboo has several secondary metabolites, the prominent among them are Cyanogenic glycosides are formed in the shoots when it is exposed to light if causes bitterness¹². For edible purpose, young bamboo shoots are used as they are soft. Prior to consumption the shoots are boiled so as to remove the glycosides which are usually toxic²². In the present study, the phytochemical composition, antioxidant, antimicrobial and antiproliferative activity of bamboo shoot extract was studied along with identification of the chemical composition of the extracts and presence of amygdalin. The results show that methanol extract of bamboo shoot have high diversity of secondary metabolites. The phenolic and flavonoids content was very high in methanol extract as compared to other extracts. Flavonoids are highly effective scavengers of most oxidizing molecules. Flavonoids suppress the formation of reactive oxygen formation, it chelate the trace elements which are involved in free radical production and also protect the antioxidant defenses²³.  The shoots also have high level of polyphenols and flavanoids which indicates that they are good for human health as they are considered as antioxidants²⁴. Phenolics compounds are having multiple biological effects, which includes scavenging oxygen. High quantity of phenolics also is the reason for high antimicrobial activity observed in methanolic extract²⁵.

 

Amygdalin is a Phytochemical which is found in high quantities in apricot. It is a major ingredient in Chinese herbal medicine ^26-27. Bamboo shoot mixed with crushed leaves of Allium Porrum and Chilli is used to treat influenza and to treat fungal infection. To cure wounds and poisonous bites concentrated liquor of boiled tender shoots of B. nutans species can be applied²⁸. Tender bamboo species of B. tulda is boiled with water and consumed in the form of soup is used to treat ulcer, poxes and other skin diseases²⁹. GC-MS analysis of methanolic showed the presence of beta.-Sitosterol; 2-Methyl-7-phenylindole, 1-(Methylamino)anthraquinone, (R)-(-)-14-Methyl-8-hexadecyn-1-ol and 2-Ethylbutyric acid, nonadecyl ester as major compounds.  Bamboo shoots in any form are a good source of phytosterols. It has lipid lowering effect³⁰. The presence of major sterols present in bamboo shoots are β- sitosterol, campestrol and stigmastrol. Phytosterol rich diets reduces colon cancer, breast cancer and Prostate cancer²⁹. In bamboo shoots there are eight major phenolic compounds are found, they are namely gallic acid, chlorogenic acid, vanillic acid, protocatechuic acid, P- hydroxybenzoic acid, syringic acid, shikimic acid and ferulic acid³¹. The species of (P. pubescens) bamboo shoot has total 20 compounds among them  two are  novel compounds such as cyclic  adenine- (1´R,2´R,3´R)-cyclic butanetetraol carbonate and (-)-(7R,8S)-(4-hydroxy-3-methoxyphenylglycerol 9-O-β-D-[6-O-4-hydroxy-3-methoxybenzoyl])-glycopyranoside³². Amygdalin is the effective component which is used in traditional Chinese medicine for the treatment of asthma, bronchitis, emphysema, leprosy, colorectal cancer and vitiligo³³. Bamboo shoots of four different species were evaluated in terms of antibacterial activity, total phenol content, flavonoid content, antioxidant activity. The antioxidant activity of methanolic extract was found to be highest among all the other extracts³⁴. Leleba oldhami Nakal bamboo shoots has show significant improved the lipid metabolism disorder situation in hyperlipidemia mice³⁵. Antimicrobial activity of acetone extracts of Bambusa vulgaris bamboo shoots on Enterococcus faecalis, Klebsiella pneumonia bacteria and Aspergillus fumigates. The shoots also have antiproliferative activity³⁶. In this study it has been observed that the antimicrobial activity of Bambusa arundinacea methanolic extract was very high by forming the zone of inhibition. Whereas MTT assay was done by using the prostate cancer cell line found that as the dose of the extract was increased the number of viable cells was decreased and the inhibition was also increased. The study claim that the shoots of  Bambusa arundinacea is useful for treating cancer and microbial invasion.

 

CONCLUSION:

The results of study shows that methanolic extract of bamboo is rich in secondary metabolites. The phytochemical analysis showed high content of phenolic and flavanoids. GC-MS analysis shows presence of 40 compounds. High antimicrobial activity was observed in methanol extracts. Antiproliferative activity was observed in PC3 cell lines. Methanol extracts can be used for development of anticancer and antimicrobial drugs besides, being a source of antioxidant.

 

ACKNOWLEDGEMENTS:

The author acknowledges Periyar University, Salem for providing the required research facilities.

 

CONFLICT OF INTEREST:

The author declares that there is no conflict of interest in publishing this manuscript.

 

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Accepted on 05.01.2021           © RJPT All right reserved

Research J. Pharm. and Tech 2021; 14(11):5661-5667.