Standardization and Phytochemical Screening analysis for Herbal Extracts: Zingiber officinalis, Rosc., Curcuma longa Linn., Cinnamonum zeylanicum Nees., Piper longum, Linn., Boerhaavia diffussa Linn.

Ruchika Sharma*, Neha Kumari, Dr. M.S Ashawat, C.P.S Verma

Sri Sai College of Pharmacy, Badhani, Pathankot.

*Corresponding Author E-mail: Ruchikasharma471@gmail.com, neha91sn@gmail.com

ABSTRACT:

The aim of the study was to standardization and phytochemical screening analysis for Herbal Extracts: Zingiber Officinalis, Rosc., Curcuma Longa Linn., Cinnamonum Zeylanicum Nees., Piperlongum, Linn., Boerhaavia Diffussa Linn. Phytochemical analysis of extracts, preformed identification tests of extracts and it is the qualitative analysis. The study involved chromatographic fingerprinting of five drug extracts; cinnamon (Cinnamomum Zeylanicum Nees), long pepper (Piper Longum, Linn.), turmeric (Curcuma Longa Linn), punarnava (Boerhaavia Diffussa, Linn.) and ginger (Zingiber Officinalis, Rosc.) for standardization followed by preparation of tablets by direct compression methods. Thin layer chromatography (TLC) and High performance liquid chromatography (HPLC) of extracts were performed and it is the quantitative and qualitative analysis. Phytochemical screening test of all five extracts; ginger, cinnamon, curcumin, long pepper and punarnava for alkaloid, carbohydrates, glycocides, cardio glycosides, saponin glycosides, tannin and steroids test were present. Thin Layer Chromatography (TLC) was performed to all extracts: Rf value 0.272 with mobile phase toulene: ethyl acetate (93:7), Rf value 0.40 with dichloromethane: methanol (60:40), Rf value 0.466 with petroleum ether: alcohol (70:30), Rf value 0.396 with toulene: ethyl acetate (93:7) and Rf value 0.333 with benzene: acetic acid (75:25) of Zingiber Officinale, Rosc., Cinnamonum Zeylanicum, Nees., Piper Longum, Linn., Curcuma Longa, Linn. and Boerhaavia Diffussa, Linn. respectively. high Performance Liquid Chromatography (HPLC) was used for quantitative purpose of all five extracts. The quantitative separation was confirmed on Shimadzu equipment. The extracts in flow rate of 1ml/min at the wavelength 282nm, 425nm, 221nm, 345nm and 233nm of Ginger, Turmeric, Cinnamom, Long Pepper and Punarnava respectively.

KEYWORDS: Phytochemical Screening, HPLC, TLC, Standardization, Extracts (Ginger, Cinnamon, Curcumin, Long Pepper and Punarnava).

INTRODUCTION:

The importance of medicinal plant in drug development is known to us and humans have used them for different diseases from the beginning of human history. Traditional folk treatment from wild plant has always guided researchers to search for novel medications to develop healthy life for humansand animals. In addition, some medicinal plants are still obscured within the plant need to be scientifically evaluated.¹

Ginger is a herbal drug used in medication as well a culinary spice and its medicinal use dates as back as to ancient China and India. Ginger is obtained from dried rhizome of Zingiber Officinale belongs to Zingiberaceae family. Indigenous to topical areas such as Asia (India and China), West Africa and Jamaica. The perennial plant is 2-4 feet tall with a green purple flower in terminal spikes.² The rhizomes of ginger are buff colored with longitudinal striations or are fibrous with no cork. An oily liquid comprising of homologous phenols called gingerol accounts for the pungency as well as the pharmacological activity of ginger.³ Ginger and its constituents such as anti emetic, carminative, stimulant, anti inflammatory, cardiotonic, antioxidant, antitussive, antibacterial, cholagogue actions as well as to promote gastric secretions, increase intestinal peristalsis, lower cholesterol levels and anti thrombotic. Ginger in high doses can cause CNS depression or cause hindrance with cardiovascular activity.⁴

The bark of various cinnamon species is one of the most important and popular spices used worldwide not only for cooking but also in traditional and modern medicines. Cinnamon consist of dried inner bark of shoots of coppiced trees of Cinnamon zeylancium Nees.⁵ Cinnamon oil contains 60-70% 0f cinnamaldehyde, 5-10% eugenol, cinnamyl alcohol, cinnamic acid, coumarin, benzaldehyde, cuminaldehyde and other terpenes like phellandrene, pinene, cymene, caryophyllene, etc. Bark issued as carminative, stomachic and mild astringent. It has been used as an expectorant and demulcent. It is also used as an antispasmodic. The major diseases for prevention and or treatment of which, nutraceutical have been associated are heart diseases, cancer, hypertension and diabetes.⁶

Turmeric is a plant that has a very long history of medicinal use, dating back nearly 4000 years. In Southest Asia, turmeric is used not only as a principal spice but also as a component in religious ceremonies. Curcumin bright yellow chemical produce by some plant. It consists of dried as well as, fresh rhizomes of the plant known as curcuma longa linn.⁷ The chief component of curcuminoids is known as curcumin (50-60%). Chemically, Curcuma species contains volatile oil, starch and curcumin. Curcumin and other related curcuminoids are reported to be responsible for the yellow color in some species. And also curcumin, curcuminoids, cinnamic, coumaric acid are chemical constituents. It contains not less than 4% of volatile oil. It is used in cosmetic products. Depression may be as effective and safe treatment for patients with major depressive disorder.⁸

The word pepper is derived from the Sanskrit word for long pepper (pippali). Long pepper (piper longum), sometime called Javanese, Indian, or Indonesian long pepper, is a flowering vine in the family Piperaceae cultivated for its fruit, which is usually driedf and used as a spice. A major source of long pepper in antiquity was the Malabar Coast where the peppercorns were traded as a source of “black gold”. It contains Piperine, Piperlongumine, rutin, beta-caryophllene piperyline, piperamine, pinene, linalool and limonene.⁹ Long pepper are effective for many diseases, including cancer, inflammation, insomnia (sleeplessness), headache, toothache, heart problems, piles, depression, diabetes, obesity and hepatotoxicity. Piper longum, sometimes called Indian long pepper (pippli), is a flowering vine, which is usually dried and used as a spice and seasoning. Long pepper is widely used to prepare vegetable pickles, for insomnia (sleeplessness), in headache, in toothache, for heart problem and for piles. Herbs is used as diuretics, Expectorant, Stomachic, Enlargement of spleen and also used for relieving abdominal pain.¹⁰

Punarnava (Hogweed) literally means ‘bring back to life’ or ‘renewer’. It is a creeper that grows wild in India and Brazil throughoutout year but dries during the summer. It bears small fleshy leaves, small reddish pink flower and fruits in winter. Punarnava consists of fresh, as well as, dried herb Boerhaavia diffussa linn belongs to Nyctaginaceae. It is found wild throughout India and Sri Lanka. Punarnava is found in Himalayan valleys upto 2000-2500 m. The weed also grows in Malaysia, China and Africa.¹¹ Punarnava contains about 0.04 – 0.1% of alkaloid known as punarnavine (M.P. 235˚C) and punernavoside, an antifibrinolytic agent. It also contains about 6% of potassium nitrate, an oily substance, and ursolic acid. punarnavine, punernavoside, b-sitosterol, palmtic acid, ursolic acid. Herb is used as diuretic, expectorant, stomachic, enlargement of spleen and used for relieving abdominal pains. Punarnava is beneficial in treating obesity; it is effective in treating a disease called dropsy.¹²

METHODOLOGY:

Drug identification:

Physical characterization⁴

Physical description of the herbal extracts ginger, turmeric, cinnamon, long pepper and punarnava was characterized by visually on the basis of colour, odour and texture.

Determination of melting point^9,10

The determination of melting point is a major identification characteristic in the pre-formulation study. The melting point and solubility are related via the latent heat of fusion process. The melting point of herbal extracts of ginger, turmeric, cinnamon, long pepper and punarnava was determined using digital melting point apparatus (Rolex, Ambala, India). Melting point of these 5 extracts was determined by capillary fusion method one sided closed capillary filled with drug and put into the digital melting point apparatus. Temperature was noted at which solid drug changed into liquid. Melting point was recorded and compared with literature.

Phytochemical analysis:

All 5 extract (ginger, cinnamon, turmeric, long pepper, punaranava) were subjected to qualitative phytochemical tests for different constituents like alkaloids, amino acids, carbohydrates, flavonoids, glycosides, cardiac glycosides, saponin glycosides, tannins, steroids and triterpenoids.

Detection of alkaloids:¹³

a) Dragendroff’s test:

Extracts were treated with Dragendroff’s reagent (solution of Potassium Bismuth Iodide) and observed for formulation of red precipitate which indicates the presence of alkaloids.

b) Wagner’s test:

Extracts were treated with Wagner’s reagent (Iodine in Potassium Iodide) and observed for formulation of reddish brown precipitate which indicates the presence of alkaloids.

c) Mayer’s test:

Filtrates were treated with Mayer’s reagent (saturated picric acid solution) and observed for presence of alkaloids confirmed by the formulation of yellow coloured precipitate.

Detection of carbohydrates:¹⁴

a) Molisch’s test:

Extracts were treated with 2 drops of alcoholic α-naphthol solution in a test tube then few drops of concentrated sulphuric acid was added through sides of the test tube and observed for formulation of the red cupric oxide which shows the presence of monosaccharide.

b) Barfoed’s test:

1ml of test solution was heated with 1ml of Barfoed’s reagent on the water bath, and observed for formulation of red cupric oxide which shows the presence of monosaccharide.

Detection of saponins glycosides:¹⁵

a) Froth formulation test: 2ml of solution of extract in water was taken in test tube and observed for the formulation of the stable foam which indicates the presence of saponins.

Detection of steroids and triterpenoids:¹⁶

a) Salkowski’s test:

Extracts were treated with few drops of concentrated sulphuric acid and observed for the red colour at the lower layer which indicates the presence of the steroids and formulation of yellow coloured lower layer which indicates the presence of the triterpenoids.

 Detection of tannins:¹⁷

a) Ferric chloride test:

Extracts were treated with ferric chloride solution and observed for the appearance of blue colour which indicates the presence of hydrolysable tannins and appearance of green colour indicates the presence of condensed tannins.

Detection of flavonoids:¹⁸

a) Alkaline reagent test:

Extracts were treated with few drops of sodium hydroxide solution and observed for formulation of intense yellow colour, which becomes colourless on addition of dilute acid which indicates the presence of flavonoids.

Detection of glycosides:¹⁹

a) General test:

The extract was treated with 5ml of dilute sulphuric acid by warming on water bath and filtered. Acid extract was then neutralized with 5% solution of sodium hydroxide. Then 0.1ml of Felhing’s solution A and B were added until it became alkaline and heated on water bath for 2 minutes and observed for the formulation of red precipitates.

Detection of cardiac glycosides:²⁰

a) Keller killani Test:

In 2ml extract, glacial acetic acid, one drop 5% (w/v) ferric chloride and conc. sulphuric acid were added. Reddish brown color appears at junction of the two liquid layers and upper layer appears bluish green, confirming the presence of glycosides.

Thin layer chromatoghraphy (TLC)²¹

After the preliminary phytochemical screening, thin layer chromatography (TLC) was preformed for all the five extracts.

a) Coating material or stationary phase:

Silica Gel G was used as the coating material for TLC.

b) Glass plates:

Glass plates of 10×5 cm were used for the application of the stationary phase.²²

c) Preparation of Thin layer in phase:

The slurry was prepared by mixing Silica Gel G and water in the particular ratio. Then the slurry was applied to the TLC plates using pouring method:

Pouring Method:

In this method, a measured amount of the slurry was put on the glass plate which was kept on a level surface. The plate was dipped back and forth to spread slurry uniformly over the surface. Thus a consistent thin layer of stationary phase was made on the glass plate.²³

d) Activation of adsorbent:

After making thin layer on plates, the liquid was removed by drying the plate for 30 minutes in air and then in an oven at 110˚C for another 30 minutes. This drying was done to make the adsorbent layer active.

e) Sample application:

For application of the sample solution, 2-5µl of a 1% solution of all five extracts was spotted on the activated glass plate using a capillary tube. The spot was kept atleast 2cm above the base of the plate.²⁴

f) Development Tank:

For the purpose of development, developing tank or chamber to hold TLC plates were used. The bottom of the chamber was covered upto nearly 1mm by the solvent. Three sides of the tank were lined with solvent impregnates paper while top was covered tightly with the lid. The development chamber was perfectly saturated with solvent vapors. Ascending chromatography was used for TLC, in which, the plate was placed in a development chamber at an angle of 45˚.²⁵

g) Solvent System (Mobile Phase):

The solvent system used for the (ginger, cinnamon, turmeric, long pepper and puarnava) TLC is toluene: ethyl acetate, dichloromethane: methanol, toluene: ethyl acetate, petroleum ether: alcohol, benzene: acetic acid.

h) Development technique:

The spotted TLC plates were placed in a development chamber saturated with vapors of the solvent system. The plates were kept in such a manner that the spotted area should not immerse in the mobile phase in the development tank. After the developer has transverse one-half to two-thirds the length of the plate, the plates were removed and dried and the position of the components were determined.²⁶

i) Detection of components:

After the development of the TLC plates, the detection of the colored spots was done visually. But for detecting colorless spots non-specific method of detection was used.²⁷

Non specific method:

By this method spots were detected by using following method.

i. Iodine chamber method: By this method the TLC plates were kept in the Iodine chamber for few minutes for the detection of the spots.

ii. UV chamber method: this method is used for the detection of the fluorescent components. In this method the TLC plates were observed under long UV wavelength and short UV wavelength.

j) Evaluation of the Chromatogram:

After detecting the separated solutes on the plate their evaluation was done using qualitative method. The TLC spots obtained by mobile phase 100%

Qualitative Evaluation:

In qualitative evaluation R_f value (Retardation factor) was calculated for identifying the spot

              Distance travelled by solute

R_f = ----------------------------------------------

               Distance travelled by solvent front

High performance liquid chromatography²⁸

Instruments and glasswares

High Performance Liquid Chromatography (Water 600), manual sampler, software Win chrome and detector (Waters 486), Column C-18 (ODS Hypersil) 150 x 4.6 mm, particle size 5µm. Measuring cylinder (500mL), beaker (100mL), pipette, volumetric flasks, sonicator, analytical balance, filter paper (pore size 0.45µ), whatman filter paper etc.

 Chemical requirements:

Acetonitrile, HPLC grade, methanol, water, formic acid, ortho phosphoric acid etc.

Selection of wavelength

The sensitivity of HPLC method that uses UV detection depends upon proper selection of detection wavelength. An ideal wavelength is the one that gives good response for the drugs that are to be detected. For the selection of wavelength 10µg/mL conc. of extracts were prepared in 50: 50 (methanol: water) and overlain spectra is obtained on UV spectrophotometer. It was found that 282nm (ginger), 226nm (cinnamon), 425nm (turmeric), 345nm (long pepper), 233nm (punarnava) wavelength was most suitable for the analysis.

Selection of chromatographic condition:

Chromatographic separation was achieved at ambient temperature on a reversed phase isocratic high performance liquid chromatography using a mobile phase consisting of methanol and water. The mobile phase was pumped at a rate of 1.2mL/min. The detector wavelength was set and run time was 10 minutes. To optimize the chromatographic conditions, the effect of chromatographic variables such as mobile phase pH and flow rate were studied. The resulting chromatograms were recorded and the chromatographic responses were measured.²⁹

Column:

For the chromatographic separation of drugs the column of Thermo scientific octadecylsilane Hypersil (ODS) C18 having length of 150 ×4.6mm, with particle size of 5µ was used.

Preparation of standard stock solution³⁰

Standard stock solution of extracts:

All extracts 10 mg was weighed and transferred to a 100 mL volumetric flask and dissolved in HPLC grade methanol: water in ratio of 50: 50. The contents were mixed and volume was made up to the mark with solvent to give a solution containing 100 µg/mL conc.

RESULT AND DISCUSSION:

Identification test

Physical characteristics of extracts:

Among the physical evaluation of variety of extracts were observed psychemicaly.

Ginger extract:

Extract was observed light brown in colour, aromatic in taste and pungent in odour.

Cinnamon extract:

Extract was observed dull yellowish brown in colour, fragnent in odour and aromatic in taste.

Turmeric extract:

Extract was observed yellow in colour, characterstics in odour and slightly bitter in taste.

Long pepper extract:

Extract was observed dark blackish brown in colour, aromatic in odour and hot and pungent in taste.

Punarnava extract:

Extract was observed light yellowish brown in colour, odourless and sweet, pungent and astringent in taste.

Melting point

The determined melting point of all five extracts was found as Ginger extract at 46˚C, Cinnamon extract at 172˚C, Turmeric extract at 180˚C, Long pepper extract at 126˚C and Punarnava extract at 160˚C. It is reported that all extracts having variable point.

Phytochemical Analysis:

All extracts: Ginger, Cinnamon, Turmeric, Long pepper, Punarnava were subjected to qualitative phytochemical tests for different constituents like alkaloids, carbohydrates, flavonoids, glycosides, cardiac glycosides, saponin glycoside, tannin, steroids and terpenoids as shown in table 3.1.

Table 3.1: Phytochemical screening of extract

Test for	Test performed	Ginger	Cinnamon	Turmeric	Long pepper	Punarnava	Remark
Alkaloids	Dragendroff’s Test	+	+	+	+	+	Alkaloids may be present
	Mayer’s Test	+	+	+	_	_
	Wagner’s Test	+	+	+	+	+
Carbohydr-ates	Molisch’s Test	_	_	_	_	+	Carbohydra-tes may be presnt and may br absent in some extracts
	Barfoed’s Test	+	_	_	+	_
Flavonoids	Alkaline reagent Test	+	_	+	+	+	Flavonoids may be present
Glycosides	Fehling A and B	+	_	_	+	+	Glycosides may be present
Cardio glycosides	Keller Killiani Test	+	+	_	_	+	Cardio glycoside may be present
Saponin glycoside	Forth formation test	+	+	+	+	+	Saponin may be present
Tannin	Ferric chloride Test	+	_	+	+	+	Tannin may be present
Steroids and Terpenoids	Salkawski Test	-	+	-	_	+	Steroids may be present

+ = present _ = absent

Table 3.2: Thin layer chromatograph of extracts.

Sr. no.	Extracts	Mobile Phase	Spot
1.	Zingiber officinale	Toulene : Ethyl Acetate (93 : 7)	Spot under U.V Rf = 0.272
2.	Cinnamonum zeylanicum	Dichloromethane : Methanol (60 : 40)	Spot under U.V Rf = 0.40
3.	Piper longum	Petroleum ether : Alcohol (70 : 30)	Spot under U.V Rf = 0.466
4.	Curcuma longa	Toulene : Ethyl Acetate (93 : 7)	Spot under U.V Rf = 0.396
5.	Boerhaavia diffussa	Benzene : Acetic Acid (75 : 25)	Spot under U.V Rf = 0.333

Thin Layer Chromatoghrapy (TLC):

Thin layer chromatography of all the extracts: Zingiber officinale, Curcuma longa, Cinnamonum zeylanicum, Piper longum, Boerhaavia diffussa are shown in a table 3.2.

High Performance Liquid Chromatography:

HPLC of ginger:

Mobile phase: Acetonitrile, phosphoric acid and methanol (55:44:1 v/v/v)

Standard solution: 50 mg of ginger extract dissolved and transfer in volumetric flask and volume made up with methanol.

Injection: 5µl.

Wavelength: 282 nm.

Flow rate: 1ml/min.

Fig 3.1: Chromatogram of Ginger extract.

HPLC of Turmeric:

Moble phase: Acetonitrile and 0.1% ortho phosphoric acid in the ratio of 50:50 v/v.

Injection: 5µl.

Flow rate: 1ml/min.

Wavelength: 425 nm.

Standard solution: 50mg of curcumin transfer in 50ml volumetric flask and volume made up with methanol.

Solution of 50, 100, 150, 200, 250, 300 µg/ml and volume made with methanol in each case.

Fig 3.2: Chromatogram of Turmeric extract.

HPLC of Cinnamon:

Mobile phase: Methanol: Acetonitrile: water (35:20:45 v/v/v)

Standard solution: 50 mg of cinnamon extract transfer in 50ml of volumetric flask and volume made up with methanol.

Injection: 10µl/ml.

Flow rate: 1ml/min.

Wavelength: 221 nm.

Fig 3.3: Chromatogram of Cinnamon extract.

HPLC of Long Pepper

Mobile phase: methanol: water (50:50 v/v)

Standard solution: 50mg of long pepper extract transfer in 50 ml of volumetric flask and volume made up with methanol and diluted to obtain 0.1%, 0.2%, 0.3%, and 0.4% of concentrations.

Injection: 20µl.

Flow rate: 1ml/min.

Wavelength: 345 nm.

Fig 3.4: Chromatogram of Long Pepper extract.

HPLC of Punarnava:

Mobile phase: Acetonitrile: orthophosphoric acid (60:40 v/v)

Standard solution: 50mg of extract dissolved in 50 ml of methanol.

Injection: 10µl.

Flow rate: 1ml/min

Wavelength: 233 nm

Fig 3.5: Chromatogram of Punarnava extract.

ACKNOWLEDGMENT:

This research was supported/partially supported by department of pharmacy, Himachal Pradesh Technical University, Hamirpur and my supervisors. I thankful to our colleagues who provided expertise that greatly assisted the research, although they may not agree with all of the interpretations provided in this paper.

The author’s would like to express my gratitude to my supervisors, who guided me throughout this project. I would also like to thank my friends and family who supported me and offered deep insight into the study.

CONCLUSION:

Ginger, Cinnamon, Turmeric, Long pepper and Punarnava are the useful for the health. Medicinal herbs and minerals have been in use for thousands of years in one form or the other under indigenous systems of medicines like Ayurveda, siddha, and Unani in addition to natural products used for their pharmaceutical actions. The herbal sample (extracts of ginger, turmeric, cinnamon, long pepper and punarnava) was gifted by Ayush herb, Nagrota Bagwan. The drug/extracts was identified by using test given in the official books, this was further confirmed by the TLC (Thin layer chromatography), HPLC (High performance liquid chromatography) and by the Infrared spectroscopy studies of the drug sample. Also indentified by performed the phytochemical screening tests and identify the medicinally active substances in herbal extracts. HPLC is found to be suitable method for the standardization and quantification of active ingredients/constituents in the herbal extracts sample. It is more sensitive and reliable method of standardization and hence gives more accurate results than other methods of standardization. HPLC method is useful validation tool for the standardization.

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Received on 06.04.2020 Modified on 08.05.2020

Accepted on 30.05.2020      ©Asian Pharma Press All Right Reserved

Asian J. Pharm. Tech.  2020; 10(3):127-133.

DOI: 10.5958/2231-5713.2020.00022.7