STANDARDIZATION OF AMRITAPRASHA GHRITA: A HERBAL GHEE BASED MEDICINAL PREPARATION

STANDARDIZATION OF AMRITAPRASHA GHRITA: A HERBAL GHEE BASED MEDICINAL PREPARATION

Saranya Sivaraj, Vijayalaxmi Mallannavar, G. R. Arun Raj ^* and U. Shailaja

Department of Kaumarabhritya, Sri Dharmasthala Manjunatheshwara College of Ayurveda and Hospital, Hassan - 573201, Karnataka, India.

ABSTRACT: Aim: To standardize Amritaprasha ghrita.  Materials and methods: Physico‐chemical studies like refractive index, specific gravity, acid value, saponification value, iodine value, determination of unsaponifiable matter, peroxide, viscosity, rancidity test and HPTLC were carried out as per the WHO guidelines, Indian Pharmacopoeia and Ayurvedic Pharmacopoeia. Conclusion: Standardization tests done on Amritaprasha ghrita helped in authenticating and ensuring the quality of the same.

Amritaprasha Ghrita, Under nutrition, karshya, Standardization, HPTLC

INTRODUCTION: Standardization is necessary to make sure the availability of a consistent product and can assure a reliable product with definite constituents ^{1 - 2}. The standardization of herbal medicines is always challenging as it medicines contain more than one active principles and the active compound is frequently unknown ^{3 - 4}. Standardization of herbal formulations is essential to assess quality, consistency of active principles and therapeutic efficacy of drugs ^{5 - 6}. The quality assessment of herbal formulations is important to justify their acceptability and safety^{7 - 8}.

Jivanti (Leptadenia reticulata (Retz.) Wight & Arn),¹⁰ Shunti (Zingiber officinale Roscoe), ¹² Shati (Hedychium spicatum Sm. in A.Rees),¹³ Shalaparni (Desmodium gangeticum (L.) DC.), ¹⁴ Prishniparni (Uraria picta (Jacq.) DC.), ¹⁵ Mudgaparni (Phaseolus trilobus Ait.), ¹⁶ Mashaparni (Teramnus labialis (L. f.) Spreng.), ¹⁶ Meda (Asparagus racemosus Willd.), ¹⁰ Mahameda (Asparagus racemosus Willd.), ¹⁰ Kakoli (Withania somnifera (L.) Dunal), ¹⁷ Ksheerakakoli (Withania somnifera (L.) Dunal), ^{18 - 19} Bruhati (Solanum indicum.), ¹⁶ Kantakari (Solanum xanthocarpum Schrad. & H. Wendl.), ¹⁴ Sweta Punarnava (Boerhavia diffusa L.), ¹⁰ Rakta Punarnava (T. portulacastrum L.), ²⁰ Madhuka (Glycyrrhiza glabra L.), ^{21 - 24} Kapikachu (Mucuna pruriens (L.) DC.), ²⁵ Shatavari (Asparagus racemosus Willd.), ¹⁰ Riddhi (D. bulbifera L.), ¹⁰ Vriddhi (Dioscorea bulbifera L.), ¹⁰ Parushaka (Grewia asiatica L.), ²⁶ Bharangi (Clerodendrum serratum. (L)), ²⁷ Mrudvika (Vitis vinifera L.), ²⁸ Shringhataka (Trapa bispinosa Roxb.), ²⁹ Tamalaki (Phyllanthus niruri L.), ¹⁰ Vidarikanda (Pueraria tuberosa (Willd.) DC), ³⁰ Pippali (Piper longum L.), ³¹ Bala (Sida cordifolia L.), ¹⁰ Badara (Ziziphus jujube Mill.), ³² Akshotaka (Juglans regia L.), ³³ Kharjura (Phoenix dactylifera L.), ³⁴ Vatama (Prunus amygdalus Batsch), ³⁵ Abhishuka (Pistacia vera L.), ³⁶ Dhatri (Phyllanthus emblica L), ³⁷ Ikshu (Saccharum officinarum L.), ³⁸ Chaaga Mamsarasa (meat soup of goat fried with ghee), ³⁹ Go ksheera, ⁴⁰ Go ghrita, ⁴¹ Madhu (honey), ⁴² Sarkara (sugar), Maricha (Piper nigrum L.), ⁴³ Twak (Cinnamomum zeylanicum Blume), ¹⁰ Ela (Elettaria cardamomum (L.) Maton), ⁴⁴ Patra (Cinnamomum zeylanicum Blume), ⁴⁵ Nagakesara (Mesua ferrea L.) ⁴⁶. Amritaprasha Ghrita is made use in the management of Karshya (Grade 1 & 2 under nutrition) in children. Literature survey did not reveal any standards for Amritaprasha ghrita and hence the current study was undertaken to standardize the same. The ingredients of Amritaprasha ghrita is detailed in Table 1.

TABLE 1: SHOWING INGREDIENTS OF AMRITAPRAASHA GHRITA

MATERIALS AND METHODS: Physico‐ chemical studies like refractive index, specific gravity, acid value, saponification value, iodine value, determination of unsaponifiable matter, peroxide, viscosity, rancidity test and HPTLC were carried out as per the WHO guidelines, Ayurvedic Pharmacopoeia and Indian Pharmacopoeia.

Plant Material: The constituents of Amritaprasha Ghrita were collected from the local market of Hassan District, Karnataka State, India in the month of March 2017. The collected drug was identified and authenticated (no: SDMCAH-DG/2017/16) at the teaching pharmacy of Department of Dravyaguna (Ayurveda Pharmacology), SDM College of Ayurveda and Hospital, Hassan, Karnataka State, India.

Methodology: The studies were done at SDM Centre for Research in Ayurveda and Allied Sciences, Kuthpady, Udupi, Karnataka State, India as per standard procedure.

Refractive Index: Placed a drop of water on the prism and adjusted the drive knob in such a way that the boundry line intersects the separatrix exactly at the centre. Noted the reading. Distilled water has a refractive index of 1.33217 at 28˚C. The difference between the reading and 1.3325 gives the error of the instrument. If the reading is less than 1.3320, the error is minus (-) then the correction is plus (+) if the reading is more, the error is plus (+) and the correction is minus (-). Refractive index of oil is determined using 1 drop of the sample. The correction if any should be applied to the measured reading to get the accurate refractive index. Refractive index of the test samples were measured at 28˚C.

Specific Gravity: Cleaned a specific gravity bottle by shaking with acetone and then with ether. Dried the bottle and noted the weight. Cooled the sample solution to room temperature. Carefully filled the specific gravity bottle with the test liquid, inserted the stopper and removed the surplus liquid. Noted the weight.  Repeated the procedure using distilled water in place of sample solution.

Acid Value: Weighed 2-10 g of ghritha in a conical flask. Added 50 ml of acid free alcohol-ether mixture (25 +25ml) previously neutralised with the 0.1M potassium hydroxide solution and shaken well. Added One ml of Phenolphthalein solution and titrated against 0.1M Potassium hydroxide solution. End point is the appearance of pale pink colour. Repeated the experiment twice to get concordant values.

Saponification Value: Weighed 2 g of the Amritaprasha ghritha into a 250 ml RB flask fitted with a reflux condenser. Added 25ml of 0.5M alcoholic potash. Refluxed on a water bath for 30 minutes. Cooled and added 1 ml of phenolphthalein solution and titrated immediately with 0.5 M Hydrochloric acid (a ml). Repeated the operation omitting the substance being examined (blank) (b ml). Repeated the experiment twice to get concordant values.

Iodine Value: The sample was accurately weighed in a dry iodine flask. Dissolved with 10 ml of CCl₄, 20 ml of iodine monochloride solution was added. Stopper was inserted, which was previously moistened with solution of potassium iodide and flask was kept in a dark place at a temperature of about 17 ºC for 30 min. 15 ml of potassium iodide and 100 ml of water was added and shaken well. This was titrated with 0.1N Sodium thiosulphate, starch was used as indicator. The number of ml of 0.1N sodium thiosulphate required (a) was noted. The experiment was repeated with the same quantities of reagents in the same manner omitting the substance. The number of ml of 0.1N sodium thiosulphate required (b) was noted. The experiment was repeated twice to get concordant values.

Determination of Unsaponifiable Matter: Weighed 5 g of the Amritaprasha ghritha into the flask. added 50 ml alcoholic KOH into the sample. Boiled gently but steadly under reflux condenser for one hour. The condensor was washed with 10ml of ethyl alcohol and the mixture was collected and transferred to a separating funnel. The transfer was completed by washing the sample with ethyl alcohol and cold water. Altogether, 50 ml of water was added to the separating funnel followed by an addition of 50 ml petroleum ether. The stopper was inserted and shaken vigorously for 1 min and allowed it to settle until both the layers were clear. The lower layer containing the soap solution was transferred to another separating funnel and repeated the ether extraction six times more using 50 ml of petroleum ether for each extraction. All the extracts were collected in a separating funnel. The combined extracts were washed in the funnel 3 times with 25 ml of aqueous alcohol and shaked vigorously. And drawing off the alcohol-water layer after each washing. The ether layer was again washed repeatedly with 25 ml of water until the water no longer turns pink on addition of a few drops of Phenolphthalein indicator solution. The ether layer was transferred to a tarred flask containing few pieces of pumice stone and evaporated to dryness on a water bath. Placed the flask in an air oven at 85 °C for about 1 h to remove the last traces of ether. A few ml of acetone was added and evaporated to dryness on a water bath. Cooled in a desicator to remove last traces of moisture and then weighed.

Peroxide  Value: 5 g of the Amritaprasha ghrita was weighed accurately into a conical flask, added 30 ml of mixture of 3 volumes of  glacial acetic acid and 2 volumes of chloroform, added 0.5 ml of potassium iodide, allowed it to stand for 1 minute, add 30 ml of water titrate gradually with vigorous shaking with 0.1M sodium thiosulphate until the yellow color disappears. Add 0.5 ml of starch indicator continued the titration until blue color disappears.

Peroxide value= 10(a-b) / W

Where W= weight in g of the substance

Viscosity: The given sample is filled in a U tube viscometer in accordance with the expected viscosity of the liquid so that the fluid level stands within 0.2 mm of the filling mark of the viscometer when the capillary is vertical and the specified temperature is attained by the test liquid. The liquid is sucked or blown to the specified height of the viscometer and the time taken for the sample to pass the two marks is measured. Viscosity is measured using the formula:

η1=    ρ1t1 X η2

ρ2t2

η1 – Viscosity of sample

η2 - Viscosity of water

t1 and t 2-  time taken for the sample and water to pass the meniscus

ρ1 and ρ2 – Density of sample and water

X= Specific gravity of sample x 0.9961/specific gravity of water

?= Xx Time for samplex1.004/specific gravity of water x70sec

Rancidity Test: 1 ml of melted fat was mixed with 1ml of conc. HCl and 1 ml of 1% solution of phloroglucinol in diethyl ether and then mixed thoroughly with the fat acid mixture. A pink color indicates that the fat is slightly oxidized while a red color indicates that the fat is definitely oxidized.

Sample Preparation for HPTLC: Sample obtained in the procedure for the determination of unsaponifiable matter is dissolved in 10 ml of chloroform this was followed for all the sample of Amritaprasha ghritha, and chloroform soluble portion was used for HPTLC.

HPTLC: 4, 8 and 12 µl of the above sample of Amritaprasha ghrita was applied on a precoated silica gel F254 on aluminum plates to a band width of 8 mm using Linomat 5 TLC applicator. The plate was developed in toluene - ethyl acetate (9:1) and the developed plates were visualized under short UV, long UV, and after derivatisation in vanillin-sulphuric acid spray reagent it was visualized under white light and scanned under UV 254 nm, 366 nm and 620 nm. R_f, colour of the spots and densitometric scan were recorded.

TABLE 2: SHOWING RESULTS OF STANDARDIZATION PARAMETERS

FIG. 1: TLC PHOTO DOCUMENTATION OF CHLORO-FORM FRACTION OF AMRITAPRASHA GHRITA

TABLE 3: R_f VALUES OF THE SAMPLE OF AMRITAPRASHA GHRITA

*F-fluorescent; D-dark; L-light

FIG. 2: DENSITOMETRIC SCAN OF AMRITAPRASHA GHRITA

RESULTS AND DISCUSSION: The standardization parameters of Amritaprasha ghrita are detailed in Table 2. The TLC photo documentation of chloroform fraction of Amritaprasha ghrita is shown in Fig. 1. The Rf values of sample of Amritaprasha ghrita is detailed in Table 3. The Densitometric Scan of Amritaprasha ghrita is shown in Fig. 2. The physicochemical standards would serve as preliminary test for the standardization of the formulation. Tests such as refractive index, specific gravity, acid value, saponification value, iodine value, determination of unsaponifiable matter, peroxide, viscosity, rancidity test and HPTLC, results of TLC photo documentation, the unique R_f values, densitometric scan and densitogram obtained at different wavelengths can be used as fingerprint to identify the herbal drug of Amritaprasha ghrita.

CONCLUSION: Amritaprasha ghrita has been standardized using diverse scientific quality parameters. The results obtained can be used as reference while setting the pharmacopoeial standards for Amritaprasha ghrita for the benefit of the end user without any unwarranted complications.

ACKNOWLEDGEMENT: Authors are highly grateful to the constant support and guidance of Dr. Prasanna N. Rao, Principal, SDM College of Ayurveda, Hassan and Dr. Mallika KJ, Research Dean, SDM College of Ayurveda, Hassan. The Authors thank Dr. B. Ravishankar, Director, SDM Centre for Research in Ayurveda and Allied Sciences for providing the facilities and guidance.

CONFLICT OF INTEREST: Nil

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    How to cite this article:

    Sivaraj S, Mallannavar V, Raj GRA and Shailaja U: Standardization of Amritaprasha ghrita: a herbal ghee based medicinal preparation. Int J Pharm Sci & Res 2018; 9(11): 4842-48. doi: 10.13040/IJPSR.0975-8232.9(11).4842-48.

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