Effect of Conventional Method and Microwave-assisted Extraction on Phytoconstituents of Operculina turpethum

Neeraj Choudhary^1,2, Ashish Suttee¹*

¹School of Pharmaceutical Sciences, Lovely Professional University, Phagwara, Punjab.

²Faculty of Pharmaceutical Sciences, PCTE Group of Institutes, Ludhiana, Punjab.

*Corresponding Author E-mail: ashish7sattee@gmail.com

ABSTRACT:

From past several years the conventional methods are generally employed in plant material extraction that include Soxhlet, reflux apparatus but the method is time consuming and requires solvent in large quantity. To overcome this problem the novel extraction technique are nowadays used for the extraction of the plant material. The Microwave assisted extraction technique offers several advantages over conventional method i.e. low solvent consumption, shortened extraction time with increased purity and yield of bioactive phytoconstituents. Operculina turpethum (Convolvulaceae) found throughout India at an altitude of about 1000m and commercially cultivated in Ceylon, tropical America, Mauritius, Philippines, Australia and tropical Africa. In India it is sometimes grown in gardens as an ornamental plant. The roots/ rhizomes of the Operculina turpethum was extracted by conventional and microwave assistant extraction using various solvents pet. ether, ethyl acetate, methanol, hydroalcholic and aqueous solvents. The result indicates that the microwave extraction process gives high yield of phytoconstituent when compared with conventional methods. The extracts obtained by both the process were further evaluated for assessment of total phenolic, flavonoid and saponin content. The research findings suggest that the microwave assisted extraction techniques has remarkably increased the phenolic, flavonoid and saponin content in the extract. Furthermore, microwave irradiation method proved to be a rapid and improved system for the plant extraction.

KEYWORDS: Operculina turpethum, Microwave Assisted Extraction, Content Estimation.

INTRODUCTION:

Traditional medicines play a key task in healing of several ailments [1] [2]. Moreover a key barrier is lack of documented evidence and lack of stringent quality control measures that restricted the use of alternative medicines in the developed countries. Moreover there is need to record the experimental work carried out on traditional system of medicines in order to develop documented evidence. With this shortcoming, it is essential to carry out the crude drug standardization in order to ensure quality in herbal medicines [3]. The conventional methods are used for the extraction of plant material that include Soxhlet and reflux apparatus but the method is time consuming and requires solvent in large quantity. The use of huge quantity solvent increases the production cost and environmental problems [4].

Therefore the need of the current era is to use novel extraction techniques that can overcome the problem associated with conventional method. In this context, Microwave assisted extraction (MAE) can enhance the extraction efficiency by producing the high-purity extracts, low solvent consumption and thereby reducing the time of extraction. The MAE is widely employed in the bioactive compounds extraction from the plant materials [5]. MAE is characterized in green technology as it decreases the consumption of organic solvent.

Operculina turpethum (Convolvulaceae) found throughout India at an altitude of about 1000 m and commercially cultivated in Ceylon, tropical America, Mauritius, Philippines, Australia and tropical Africa. In India it is sometimes grown in gardens as an ornamental plant [6][7]. The Operculina turpethum commonly recognized as Nisoth which is widely distributed across the globe that contains various phytoconstituents which are helpful in the treatment of several diseases. Therefore in the current research an attempt has been made to explore the effect of conventional and MAE on the phytoconstituents of the Operculina turpethum.

MATERIALS AND METHODS:

Collection and authentication of plant material

The roots/ rhizomes of Operculina turpethum was collected in the month of June 2018 from local areas of Ludhiana (Punjab) and authenticated by Dr. Sunita Garg, Emeritus Scientist, Department of Raw Material Herbarium & Museum, NISCAIR, New Delhi having voucher specimen number Ref. No. NISCAIR/RHMD/ Consult/2018/3227/28-2.

Conventional Method:

After collection the plant materials were shade dried at room temperature, coarsely crushed by a pulveriser. The powdered plant materials were successively extracted in a Soxhlet apparatus using pet. ether, ethyl acetate, methanol, hydroalcholic and aqueous solvents to get respective extracts. The solvent was removed by distillation and concentrated in vacuum in a rotary evaporator. All the dried extracts were placed in desiccators till further use [8].

Microwave Assisted Extraction:

The roots/ rhizomes of Operculina turpethum was extracted using different solvent i.e. pet. ether, ethyl acetate, methanol, hydroalcholic and aqueous solvent at 200W by means of magnetic shaker and at 50ºC temperature for 18 min [9]. The extraction was performed in triplicate and further the results for both the extraction process i.e. conventional method and MAE was compared with each other in Table 1.

Phytochemical quantification of plant extracts

Estimation of total phenolic content

Principle

The phenolic contents was estimated by Folin Ciocalteu’s method. The Folin Ciocalteu reagent is a combination of phosphotungstate and phosphomolybdate that are employed in colorimetric assay of polyphenolic and phenolic antioxidants. It helps in identification of the required substance amount which is to be tested to inhibit the oxidation of reagent. The oxidation of diluted sample extract was carried out by Folin Ciocalteu reagent and the reaction mixture was neutralized by addition of sodium carbonate. The blue colour solution absorbance was measured at 765nm after 30 min.

Preparation of Sample:

The roots/rhizomes of Operculina turpethum (1gm) were separately extracted with ethyl acetate, methanol and hydroalcholic solvent and then filter the solution adjust the volume in volumetric flask up to 50ml. 1ml of sample was mixed with distilled water (10ml) in test tube. Then add 1.5ml Folin Ciocalteu’s reagents and allowed for incubation at room temperature for 5 minutes. Add 20% (w/v) sodium carbonate (4ml). The volume of the solution was adjusted by distilled water up to 25ml, stirred well and placed at room temperature for 30 minutes. The absorbance of the solution was recorded at 765nm by UV-spectrophotometer [10]. All the results were performed in triplicate. Quantification of sample was determined by Gallic acid standard curve. The Gallic acid standard curve was shown in Figure 1 and the extract total phenolic content was shown in Table 2.

Calculation:

C= c × V /M

C-Total Phenolic content

c-Gallic acid conc. achieved by standard curve (mg/ml)

V-Extract Volume (ml)

M-Extract Mass (g)

Estimation of total flavonoid content in the plant extracts

Principle

The modified procedure of aluminum chloride colorimetric assay reported by Woisky and Salatino. The complex (acid stable) was formed due to aluminum chloride interactions with keto group C-4 position or with the C3 and C5 of flavonols and flavones hydroxyl group. Furthermore the aluminum chloride has the ability to form ortho-dihydroxyl (acid labile complex) of flavanoids.

Preparation of sample:

The roots/rhizomes of Operculina turpethum (1gm) were separately extracted with ethyl acetate, methanol and hydroalcholic solvent and then filter the solution adjust the volume in volumetric flask up to 50ml. An amount of 0.6mL sample solution mixed with 2% aluminum chloride (0.6mL). The resulting solution was further incubated for 1hr. at room temp. Then reaction mixture absorbance was recorded at 420nm by means of UV- spectrophotometer [11]. All the results were performed in triplicate. The sample quantification was carried out as per the quercetin standard curve. The quercetin standard curve was represented in Figure 2 and the total flavonoid content of extract was shown in Table 3.

Calculation

C= c × V/M

C - Total Flavonoid content

c-Quercetin conc. achieved by standard curve (mg/ml)

V-Extract Volume (ml)

M-Extract Mass (g)

Determination of total Saponins content in plant extracts:

Sample:

The roots/rhizomes of Operculina turpethum (1gm) were separately extracted with methanol and hydroalcholic solvent and then filter the solution adjust the volume in volumetric flask up to 50ml. To 1ml of sample solution 0.25ml vanillin reagent (8%,) and 2.5 ml sulphuric acid (72% v/v) were gradually added. The resulting solution was mixed and kept at 60ᵒC in water bath for 10 min and further the solution was placed for 3-4 minutes in ice water bath. The absorbance was recorded at 544nm by UV–spectrophotometer [12]. All the determinations were performed in triplicate. The sample quantification was carried out as per the diosgenin standard curve. The diosgenin standard curve was represented in Figure 3 and total saponin content in Table 4.

Calculation

C = c × V/M

C-Total saponin content

c-Diosgenin conc. achieved by standard curve (mg/ml)

V-Extract Volume (ml)

M-Extract Mass (g)

RESULTS:

Extraction

The yield of different plant extract of Operculina turpethum was increased when compared with microwave assistant extraction. The results indicate that the MAE is better in comparison with the conventional method of extraction.

Estimation of Total Phenolic content

Figure 1: Standard curve of Gallic acid

Table 2: Total phenolic contents of Operculina turpethum

S.No	Test Sample (Operculina turpethum)	Conventional extraction total phenolic content in mg/g equivalent of Gallic acid	MAE total phenolic content in mg/g equivalent of Gallic acid
1	Ethyl acetate Extract	1.20 ± 0.026	2.08 ± 0.068
2	Methanol Extract	2.52 ± 0.026	3.71 ± 0.045
3	Hydro alcoholic Extract	1.26 ± 0.045	2.08 ± 0.068

Estimation of total flavonoid content

Figure 2: Standard Curve of Quercetin

Table 3: Total flavonoids contents of Operculina turpethum

S.No	Test Sample (Operculina turpethum)	Conventional extraction total flavonoids content in mg/g equivalent of quercetin	MAE total flavonoids content in mg/g equivalent of quercetin
1	Ethyl acetate Extract	1.89 ± 0.039	2.60 ± 0.029
2	Methanol Extract	3.30 ± 0.038	4.15 ± 0.047
3	Hydro alcoholic Extract	1.07 ± 0.047	1.67 ± 0.029

Estimation of total saponin content

Figure 3: Standard curve of Diosgenein

Table 4: Total saponin contents of Operculina turpethum

S. No

Test Sample (Operculina turpethum)

Conventional extraction total saponin content in mg/g

equivalent of diosgenin

MAE total saponin content in mg/g

equivalent of diosgenin

Methanol Extract

2.87 ± 0.061

3.79 ± 0.106

Hydro alcoholic Extract

1.77 ± 0.106

3.33 ± 0.061

CONCLUSION:

The research findings suggest that the MAE techniques has significantly increases the yield and percentage of phytoconstituents i.e. phenolic, flavonoid and saponin content in the extracts of the plant Operculina turpethum in comparison with conventional method of extraction.

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Received on 24.03.2020 Modified on 13.05.2020

Research J. Pharm. and Tech. 2020; 13(12):6109-6112.

DOI: 10.5958/0974-360X.2020.01065.3

Extract	Color observed in Day light	Consistency	Conventional Method Yield (%w/w)	MAE Yield (%w/w)
Petroleum ether extract	Light Brown	Semisolid	2.40	5.70
Ethyl acetate extract	Yellowish brown	Semisolid	8.60	13.45
Methanol extract	Reddish brown	Semisolid	11.03	17.67
Hydro alcoholic extract	Light reddish brown	Semisolid	7.35	12.89
Aqueous extract	Dark brown	Semisolid	9.15	16.91