INTRODUCTION:

Curculigo orchiodes Gaertn. belongs to Amaryllidaceae family is also known as Golden eye grass or Black Musale in English, ‘‘musali’’ or ‘‘talamuli’’ in Ayurveda, ‘‘nilapanai’’ in Siddha systems of medicine and Kalimusli in Hindi^1,2.. The rhizome of C.orchiodes when mixed with honey and applied on face, it lightness up the skin³. Its tuberous rootstock is used as a rejuvenator and aphrodisiac drug, and it is used to treat various diseases such as asthma, piles, jaundice, urinary disorders, impotence, leucorrhoea, menorrhagia and diarrhea⁴. Pharmacological studies revealed that complete plant of C.orchiodes used to treat several disorders such as immunostimulant, hepatoprotective, anticonvulsant, sedative, anticancer, antibacterial, antiosteoporotic, and hypoglycaemic^5-9.

Curculigenin A and curculigo have antihepatotoxic properties, and it is used as a yang tonifying herb for thyroidectomized rabbits^10,11. The plant flavanone glycoside is a stimulant to uterine and kidneys in guinea pigs, rats, and rabbits. It is also a stimulant to the kidneys. It can be used as an antiaging medication and form healthy food supplement according to ayurvedic formulations. Majorly, the chemical structure of the agents has higher plant secondary metabolites^12,13. Plant extracts have bioactive compounds and used as cosmetics, dyes, food additives and fine chemicals^14,15. The hexane extract contains alkaloid lycopene and three steroids such as sitosterol, stigmasterol¹⁶ and yuccagenin¹⁷. C.orchiodes isolated five phenolic compounds namely - curculigine A, orcinol glucoside, curculigoside (5- hydroxy-2-O-D-glucopyranosyl benzyl-2, 6-dimethoxy benzoate), corchioside A17 and flavanone glycoside–I (glycoside–5, 7- dimethoxy-dihydromyricetin- 3-O-L-xylopyranosyl-(4-1)- glycopyranoside)^18-20. There is a huge phytochemical compound that has been reported such as orcinolglucoside, curculigoside B, curculigoside C, 2,6-dimethoxy benzoic acid, esters, heptadecanoic acids, Tetradecanal, Cyclohexasiloxane, dodecamethyl, protoanemonine, 25-hydroxy-33-methylpentatriconta-6-one, 21-hydroxy-tetracontan-20-one and palmitic acids^21-23. The plant extracts are efficient in antimicrobial properties, in which gram-positive bacteria treat food poisoning and wound healing. Gram-negative bacteria are responsible for treating lower urinary tract infections. Developing antimicrobial properties from plants leads to the development of medicines against bacteria^24,25. Hence, the present study carried out to know the secondary metabolites present in the ethanol and methanol extracts of Curculigo orchiodes. And to investigate the antibacterial activity of different extracts of this plant.

MATERIALS AND METHODS:

Collection of plants:

The plant material was collected from the local market, Avadi, Chennai. It was washed in running tap water to remove adhering material, and shade dried at RT for a week. The dried plant materials were blended into a fine coarse powder and stored in an airtight container.

Materials:

Chemicals used for the study were purchased commercially and used without any further purification. The list of chemicals is: Ethanol, Methanol, Muller Hinton agar, Millipore water.

Preparation of plant extract:

The coarse powder of the C.orchiodes plant was successfully extracted with ethanol and methanol solvents in the ratio of 1:5 and kept in an orbital shaker for 48 hrs. The ethanol extract was filtered using Whatman No.1 filter paper and it was reciprocated for methanol extract. And the extract were dried by placing the sample in the distillation unit²⁶. The separated extract were used to screen the phytochemical compounds, to check antibacterial efficacy, and GC-MS analysis to find out the pharmacological compounds.

Phytochemical screening:

The C.orchiodes extracts were subjected to analyze the quality of secondary metabolites such as flavonoids, Tannins, Saponins, Terpenoids, Phenol, Protein, carbohydrate and cardiac glycosides which is responsible for disease recovery by the following standard protocol^{27, 28}.

Antibacterial activity of C.orchioides:

The methanol and ethanol extracts were tested for antibacterial activity in a well diffusion method. Three bacterial species namely, E. coli, Pseudomonas aeruginosa, Klebsiella pneumoniae were spread plated on to Muller Hinton agar plates. Four wells were bored in the plate with a diameter of 0.5cm and 90μL of the plant extracts were added in the bored wells. The plates were then incubated at 37°C for 24 hours. The observation was done based on different antibiotics and the zone of inhibition was measured using the scale around the discs^29,30.

GC-MS Analysis:

Bioactive compounds of methanol and ethanol extracts were determined by GCMS analysis (model – GCMS-QP 2010 plus). For GC-MS detection, an electron ionization system with ionizing energy of 70 eV was used. Helium gas (99.999%) was used as the carrier gas at constant flow rate 1mL min^-1 and an injection volume of 2μL was employed (split ratio of 1:10); Injector temperature 280°C; Ion-source temperature 250°C. The oven temperature was programmed from 50°C (isothermal for 1 min), with an increase of 10°C. Min-1, to 200°C, ending with a 5 min isothermal at 300°C. Mass spectra were taken at 70 eV in a scan-interval of 0.30 seconds. The identification of each compound was compared by their relative retention time and mass spectra. GCMS interpretation was carried out by NIST (National Institute Standard and Technology) database having 62000 patterns. The unknown compounds were compared with the NIST library is known compounds and its name, molecular mass, molecular weight, and chemical structure have been ascertained. Based on the GCMS analysis, the peak in ethanol and methanol extracts of the C.orchiodes plant showed the presence of bioactive secondary metabolites³¹.

RESULTS AND DISCUSSION:

Phytochemical screenings of the extract were revealed the presence of flavonoids, tannins, phenol, protein, cardiac glycosides, terpenoids, and carbohydrate in the ethanol and methanol extract (Table-1). Based on this analysis, ethanol extract shows the best inference of phytochemicals comparably to methanolic extract. Moreover, the ethanolic extract has anticonvulsant, sedative and adaptive effects such as hyperthermia and hypoxia³².

Table 1: Phytochemical compounds in the ethanol and methanol extract of C.orchiodes

S. No	Phytochemical Components	Ethanolic extract of C.orchiodes	Methanolic extract of C.orchiodes
1	Flavonoids	++	+
2	Saponins	-	-
3	Tannins	++	+
4	Phenol	++	++
5	Protein	+	+
6	Cardiac glycosides	+	++
7	Terpenoids	++	+
8	Carbohydrate	+	+

The results of the antimicrobial activity of ethanol and methanol extract were tested on three organisms, namely, E.coli, K.pneumonia, P.aeruginosa (Table-2). The zone of inhibition was noted around the well. The ethanol and methanol extract results were compared with the standard antibiotics such as streptomycin and penicillin. Ethanol extracts show the highest zone of inhibition against P.aeruginosa (5.28±0.350), K.pneumonia (3.4±0.200), and also compared to the standard antibiotics (1.8 to 4.8mm). Based on the inhibition of bacterial growth by the plant extracts due to the presence of active metabolites, either can act alone or inhibit bacterial growth by the combination level^33,34. Gram-negative bacteria E.coli responsible for lower urinary tract infections and septicemia, as well, methanol extract of C.orchiodes known to have hepatoprotective based on different liver marker enzymes such as alkaline phosphatase, alanine aminotransferase, and gamma-glutamyl transpeptidase.

Table 3. Phytochemical compounds identified in the ethanol extract of C.orchiodes

S. No.	Retention Time	Name of the compound	Molecular Formula	Molecular weight	Chemical structure
1	5.019	2-FURANMETHANOL	C₅H₆O₂	98
2	6.346	CYCLOPENTANONE, 2-METHYL-	C₆H₁₀O	98
3	8.072	FORMIC ACID, 2-PROPENYL ESTER	C₄H₆O₂	86
4	8.252	2,5-FURANDIONE, 3-METHYL	C₅H₄O₃	112
5	13.053	PHENOL, 3-METHOXY-	C₇H₈O₂	124
6	14.372	2-Methoxy-4-vinyl phenol	C₉H₁₀O₂	150
7	17.372	1,2-Benzenediol, 3-methoxy-	C₇H₈O₃	140
8	24.958	n-Hexadecanoic acid	C₁₆H₃₂O₂	256
9	26.777	Phytol	C₂₀H₄₀O	296

Table 4: Phytochemical compounds identified in the methanol extract of C.orchiodes

S. No.	Retention Time	Name of the compound	Molecular Formula	Molecular weight	Chemical structure
1	13.042	5-Hydroxymethylfurfural	C₆H₆O₃	126
2	14.344	2-Methoxy-4-vinyl phenol	C₉H₁₀O₂	150
3	14.506	1,4-BENZENEDIOL	C₆H₆O₂	110
4	15.015	PHENOL, 2,6-DIMETHOXY-	C₈H₁₀O₃	154
5	24.910	n-Hexadecanoic acid	C₁₆H₃₂O₂	256
6	31.771	Di-n-octyl phthalate	C₂₄H₃₈O₄	390

Table 5: Bioactive compounds identified from the ethanol extract of C.orchiodes

S. No	Phytochemical Compound	Pharmacological Actions
1	2-Furanmethanol	Antiviral and Antifungal
2	Cyclopentanone, 2-methyl-	Antibacterial
3	Formic acid, 2-propenyl ester	Antifungal
4	2,5-Furandione, 3-methyl	Anticancer
5	Phenol, 3-methoxy-	Antibacterial
6	2-Methoxy-4-vinyl phenol	Antimicrobial
7	1,2-Benzenediol, 3-methoxy-	Antioxidant
8	N-Hexadecanoic acid	Anti-inflammatory, Antioxidant and antimicrobial activity
9	Phytol	Antibacterial

Table 6: Bioactive compounds identified from the methanol extract of C.orchiodes

S. No	Phytochemical Compound	Pharmacological Actions
1	5-Hydroxymethylfurfural	Antioxidant and Anti proliferative activity
2	2-Methoxy-4-vinyl phenol	Antioxidant and Antimicrobial activity
3	1,4-Benzenediol	Antimicrobial and Anti-inflammatory
4	Phenol, 2,6-dimethoxy-	Antimicrobial activity
5	n-Hexadecanoic acid	Antimicrobial activity
6	Di-n-octyl phthalate	Antioxidant and antimicrobial activity

GC-MS analysis of C.orchiodes extract was useful for analyzing the best extracts and medicinal values of the plant. Earlier, methanolic peel extract of the Punica grantanum has been reported in the presence of 2,5 furandione 3- methyl compound, which has been reported for anticancer activity³⁵. Similarly, extract of Bunchosia armica found to have 2, furan methanol responsible for antioxidant and antiviral activity³⁶. 2-methoxy 4- vinyl phenol shows the largest peaks in the ethanol extract of Zanthoxylum Tetraspermum Wight and the compound was used as antioxidant, anti-microbial and anti-inflammatory property³⁷. Presence of Formic acid 2- propenyl ester was potential for antifungal activity of Myco-molecules of Coprinopsis cinerea against Fusarium spp.³⁸. Ethanol extract of the Kigelia pinnata and Melissa officinalis leaves were reported for the presence of Hexadenoic acid Compound^39,40. And, n-Hexadecanoic acid and Octadecanoic acid was identified as the major compounds among the 17 compounds in the leaves of Cleistanthus collinus⁴¹. The Chloroform extract of the Ehretia serrata showed the di-n-octyl phthalate compound can render activity to kill all the microorganisms^42.

The compound phytol was also observed in the leaves of Mimosa pudica⁴³, it is acrylic key diterpene alcohol that is a precursor for vitamins E and K1. In candies, it is used as a hardener with sugar or corn syrup. This component was observed to have antibacterial activities against Staphylococcus aureus which causes damage to the cell membrane; as a result, there is a leakage of potassium ions from bacterial cells. Similarly, the presence of phytol and Phenol, 2, 4-bis (1-phenylmethyl) has medicinal values as potential anticancer and antioxidant activity⁴⁴.

Based on the GC-MS result, nine compounds were identified by the ethanol extracts with their medicinal value, and it gives a higher value of phytocompounds compare to the six compounds of methanol extracts.

CONCLUSION:

This present study revealed that the analysis of two different extracts ethanol and methanol suggested the presence of 15 phytochemical compounds by GCMS analysis. These components have a broad spectrum of medicinal values like antimicrobial activity. The phytochemical analysis of C.orchiodes reveals the presence of flavonoids, tannins, phenol, protein, cardiac glycosides, terpenoids, and carbohydrates. And the ethanol extract shows a higher zone of inhibition in the antibacterial activity, more potential to kill against the tested microorganisms. Therefore, both the extract of Curculigo orchiodes can be postulated as a bioactive compound and exploited for the different medicinal activity.

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Received on 25.08.2020 Modified on 27.09.2020

Research J. Pharm. and Tech. 2021; 14(8):4355-4360.

DOI: 10.52711/0974-360X.2021.00756

	Ethanolic extract of C.orchiodes	Methanolic extract of C.orchiodes	Streptomycin	Ampicillin
Escherichia Coli	2.2±0.150	2.3±0.200	1.8±0.250	2.1±0.150
Klebsiella Pneumonia	3.4±0.200	2.8±0.350	2.51±0.100	3.2±0.200
Pseudomonas aeruginosa	5.28±0.350	4.1±0.500	4.8±0.300	4.4±0.250