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Journal of Drug Delivery and Therapeutics

Open Access to Pharmaceutical and Medical Research

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Open Access  Full Text Article                                                                                      Research Article

Qualitative Phytochemical Investigation and Acute Oral Toxicity Study on Ethanolic Extract of Curcuma longa Leaves

Pratima Chourasiya ¹*, Rekha Gour ², Anant K. Patel ³

¹ PG Student, Swami Vivekanand College of Pharmacy, Indore (M. P.), India

² Professor, Swami Vivekanand College of Pharmacy, Indore (M. P.), India

³ Professor, Swami Vivekanand College of Pharmacy, Indore (M. P.), India

 

 

INTRODUCTION

Toxicology, the foundational study of poisons, is crucial for understanding the effects of substances on living organisms. The Organization for Economic and Development (OECD) defines acute toxicity as the rapid onset of adverse effects resulting from the oral administration of a single or multiple doses within 24 hours. This field, akin to both science and art, parallels medicine in its approach, involving the collection and utilization of observational data to predict outcomes of substance exposure in both humans and animals. Throughout history, humans have classified certain plants as either harmful or safe. Recent attention has shifted towards traditional herbal therapies, such as Ayurveda Medicines, presumed to have fewer side effects compared to allopathic drugs. Consequently, there is a significant focus on identifying plants devoid of toxicity for safe human consumption. ¹

At the core of turmeric's bioactive components lies the flavonoid group known as Curcuminoids, consisting of curcumin (diferuloylmethane), demethoxy curcumin, and bisdemethoxy curcumin. Among these, curcumin constitutes the major portion, accounting for roughly 90% of the curcuminoid content in turmeric. Turmeric also comprises sugars, proteins, and resins. The extensively researched component is curcumin, which constitutes 0.3-5.4% of raw turmeric.²Curcuminoids, encompassing curcumin (diferuloylmethane), demethoxy curcumin, and bisdemethoxy curcumin, constitute the essence of turmeric. Alongside these compounds, turmeric contains volatile elements such as tumerone, atlantone, and zingiberone, in addition to sugars, proteins, and resins. Curcumin, a lipophilic polyphenol, exhibits minimal solubility in water but displays a notable level of stability within the acidic pH environment of the stomach.³

Figure 1: Curcuma Longa Plant.

MATERIALS AND METHODS 

Selection and Collection of plant materials 

Based on a review of literature and traditional assertions in different communities, the selection of Curcuma Longa L. was made for the present study. The plant material, specifically the leaves, was gathered from the medicinal garden located at Swami Vivekanand College of Pharmacy.

Collection and authentication of plant material 

The leaves of Curcuma Longa L. (Zingiberaceae) were obtained from the medicinal garden at Swami Vivekanand College of Pharmacy in Indore, Madhya Pradesh, India. Taxonomic identification was conducted at the Department of Botany, Janata PG College, A.P.S. University, Rewa (486001), M.P., India, under the supervision of Dr. S.N. Dwivedi. The reference voucher specimen number is J/Bot./2023-0118, dated 20/09/2023.

Preparation of Extract 

The Maceration method was employed for the extraction process. Fresh leaves were collected, thoroughly washed to remove soil and impurities, and then air-dried for 20 days. Subsequently, 60.52 grams of the air-dried leaf powder of Curcuma Longa was soaked in 96% ethanol solvent for 24 hours, filtered to obtain the first macerate, and then immersed again in 96% ethanol for another macerate. The two macerates were combined and poured into a porcelain evaporating dish. The resulting extract was concentrated on a boiling water bath until a semi-solid mass was achieved. The residue obtained was dried, weighed (15.67 gm), stored in a cool, dry place, and used for further experiments. The percentage yield was calculated using the following formula.⁴

The ethanolic extract was prepared by Maceration extraction technique and the % yield was calculated, its physical characteristics are tabulated as below.

 

 

Table1: Percentage extractive value and physical characteristics of extract

Figure 2: Dried Leaves

Figure 3: Extract concentrated on boiling water bath

 

 

Qualitative phytochemical analysis ^{5, 6}

The phytochemical screening of aqueous extract was done to identify the secondary metabolites present in the aqueous extract of Curcuma LongaL.using different chemical reagents.Tests were performed for the presence determination of Alkaloids, Flavonoids, Saponins, Carbohydrates, Tannins, Proteins and Terpenoids etc. Phytochemical screening was performed using standard procedure. 

I. Detection of Alkaloids: -  

a) Dragendroff’s Test: 2 ml of ethanolic extract was taken and few drops of Dragendroff’s reagent was added to it.  Formation of orange brown precipitate indicated the presence of alkaloids.

b) Mayer’s Test: 2 ml of ethanolic extract was taken and few drops of Mayer’s reagent (Potassium mercuric iodide) was added to it. Formation of precipitate indicated the presence of alkaloids.

c) Wagner’s Test: 2 ml of ethanolic extract was taken and few drops of Wagner’s reagent was added to it.  Formation of reddish-brown precipitate indicated the presence of alkaloids.

d) Hager’s Test: 2 ml of ethanolic extract was taken and few drops of Hager’s reagent was added to it.  Formation of yellow precipitate indicated the presence of alkaloids.

II.Detection of Flavonoids: -   

a) Lead acetate Test: - Extracts were treated with few drops of lead acetate solution. Formation of yellow colour precipitate indicates the presence of flavonoids.

b) Sulphuric acid Test: Extracts were treated with few drops of sulphuric acid. Formation of brownish red colour precipitate indicates the presence of flavonoids.

c) Zinc Test: Extracts were treated with zinc in the presence of heat. Formation of red colour indicated the presence of flavonoids. 

III. Detection of Tannins: -

a) Nitric acid test: Extract were treated with dilute nitric acid. Appearance of deep red colour indicated the presence of Tannins.

b) Ferric chloride Test: Extract were treated with Ferric chloride solution. Appearance of deep blue colour indicated the presence of Tannins.

IV. Test for Saponins: - 

a) Froth Test: Extracts were diluted with distilled water to 20ml and this was shaken in a graduated cylinder for 15 minutes. There is no formation of foam which indicates the absence of saponins.

b) Foam Test: 0.5 gm of extract was shaken with 2 ml of water. There is no formation of foam which indicates the absence of saponins.

V. Test for Terpenoids

a) Salkowski Test: 5 ml of ethanolic extract were taken and 2 ml of Chloroform was added to it. Further, 3 ml of concentrated sulphuric acid was added to it. Formation of reddish-brown coloured layer at the interface was formed which indicated the presence of terpenoids.

VI. Test for Glycosides

a) Legal’s Test: 2 ml of ethanolic extract was taken and 1 ml of pyridine and 1 ml of sodium nitroprusside was added to the extract. Yellow colour indicated the presence of glycosides.

b) Baljet’s Test: 2 ml of ethanolic extract was taken and 1 ml of sodium picrate solution was added to it. Formation of yellowish orange colour indicated the presence of glycosides.

VII.  Test for Volatile oils

a) Odour test: The Curcuma Longa ethanolic extract possess characteristics volatile oils. 

b) Filter paper test: The ethanolic extract has permanently removed the stains on filter paper, indicating the presence of volatile oils.

 

 

Table 2: Qualitative phytochemical analysis of ethanolic Curcuma Longa extract.

 

 

 

Selection of animals: 

In acute toxicity studies per the literature survey Swiss albino mice are used as experimental model because they are physiologically very much related to human. 

Experimental Animals 

The experimental protocol was approved by the Animal Ethical Committee of Pinnacle Biomedical Research Institutional, (Approval No: 1824/PO/ERe/S/15/CPCSEA) and were strictly in accordance with the norms of CPCSEA. New Delhi (CPCSEA stands for Committee for the Purpose of Control & Supervision of Experiments on Animals, India).

Healthy Swiss albino miceweighing 25-30g of either sex was obtained from the animal house of Pinnacle Biomedical Research Institutional, Bhopal for the evaluation of analgesics and anti-inflammatory activity. The animals were housed in well-ventilated standard polypropylene cages at controlled temperature (22ºC3ºC) and relative humidity ranging between 50-70%. The animals had 12 hrs light: 12 hrs dark cycle. The animals were kept individually in the large spacious hygenic cages during the course of experimental period.  The animals were allowed free access to standard laboratory pellets and drinking water ad libitum. The animals were allowed to acclimatize for seven days before being used for the studies. 

Chemicals and reagents used: Ethanol& for screening of the phytochemicals, the reagents and the chemicals used was of laboratory were of analytical grade 

Preparation of doses:

The ethanolic extract of Curcuma Longa and the standard drug was administered in a constant volume over range of doses to be tested by varying the concentration of dosing preparation. The test animal's size determined the maximum amount of liquid that could be given at once. The volume in rats was typically not more than 1 millilitre per 100g of body weight. The dilutions of the ethanolic extract and standard drug was prepared in normal saline acting as a vehicle. The freshly prepared doses were administered to the rats to avoid stability issues.⁷

Acute toxicity test (LD50) the Lokes method ⁸

Laboratory animals used in the research: The animal models involved in this study was Swiss albino mice. The parameters evaluated during this were assayed including body weight of the mice, survival time, paw licking, salivation, stretching/writhing, erect fur, calmness, reduced movement, weakness, coma, convulsion, sleep and death. Albino mice were coded, weighed and randomized into three groups/dose with 3 mice per group/dose in two phases. In phase I, nine mice were randomized into three groups of three mice each and were given (5 mL) each of 30, 300 and 500 mg/ kg body weight (b. wt) of the extract orally. The mice were observed for paw licking, salivation, stretching of the entire body, weakness, sleep, respiratory distress, coma and death in the first four (4) hours andsubsequently daily for seven (7) days. In phase II, another fresh set of nine mice were randomized into three groups of three mice each and were given 1600, 2900 and 5000 mg kg-1 body weight of the extract orally on the basis of the result obtained during the first phase. They were observed for signs of toxicity and mortality for the first four critical hours and thereafter, daily for 7 days. 

The LD50 was then calculated as the square root of the product of the lowest lethal dose and highest nonlethal dose i.e., the geometric mean of the consecutive doses for which 0% and 100% survival rates were recorded in the second phase, the oral median lethal dose were calculated using the formula: 

Oral median lethal (LD50) dose=√Minimum toxic dose × maximum toxic dose

 

 

Table 3: Effect of Curcuma Longa on body weight

 

 

RESULTS

Acute toxicity test (LD50) the Lokes method 

The acute toxicity tests were conducted in two phases using the ethanolic extract of Curcuma longa leaves. In the first phase, doses of 30 mg/kg (3 mg/mL), 300 mg/kg (30 mg/mL), and 500 mg/kg (50 mg/mL) were administered to three mice each. No signs of toxicity were observed, and all mice survived in each dose group.

In the second phase, higher doses of 1600 mg/kg (160 mg/mL), 2900 mg/kg (290 mg/mL), and 5000 mg/kg (500 mg/mL) were administered. Mice in the 1600 mg/kg group showed signs of weakness, Salivation, and reduced movement, but all survived. In the 2900 mg/kg group, one mouse experienced convulsions, another died, and the third exhibited sleep-like symptoms. In the 5000 mg/kg group, all three mice died. These results indicate that the ethanolic extract demonstrated toxicity at higher doses in the second phase, with lethal effects observed at 5000 mg/kg.

 

 

Table 4: Phase I Acute Toxicity Test of Ethanolic Extract of leaf of Curcuma Longa

 

Table 5: Phase II Acute Toxicity Test of Ethanolic Extract of leaf of Curcuma Longa

 

 

Oral median lethal (LD50) dose=√Minimum toxic dose × maximum toxic dose

Minimum toxic dose: 1600 mg/Kg (160 mg/mL)

Maximum toxic dose: 2900 mg/Kg (290 mg/mL)

(LD50) dose=√1600×2900

= 2,154.06 mg/kg

Low Dose: 200 mg/Kg (20 mg/mL)

High Dose: 400 mg/kg (40 mg/mL)

DISCUSSION

The acute toxicity evaluation of Curcuma longa leaves revealed noteworthy insights into its safety profile. The OECD definition of acute toxicity emphasizes the rapid onset of adverse effects following substance administration within a short duration. In our study, the ethanolic extract exhibited no signs of toxicity at lower doses (30 mg/kg, 300 mg/kg, and 500 mg/kg) during the first phase, suggesting a favorable safety profile within this dosage range.

However, the second phase, involving higher doses (1600 mg/kg, 2900 mg/kg, and 5000 mg/kg), uncovered dose-dependent toxicological effects. At 1600 mg/kg, mice displayed weakness, salivation, and reduced movement, yet all survived. The 2900 mg/kg group witnessed convulsions, one fatality, and sleep-like symptoms, while all three mice succumbed to the 5000 mg/kg dose. These outcomes underscore a dose-related toxicity pattern, reinforcing the importance of dosage considerations in the utilization of Curcuma longa extract.

The LD50, a crucial metric in toxicity assessment, was determined as 2154.06 mg/kg. This value serves as a reference point for estimating the median lethal dose, providing valuable information for future applications and regulatory considerations. The LD50 establishes a threshold beyond which adverse effects become more pronounced, guiding safe dosage determination.

CONCLUSION

In conclusion, the acute toxicity evaluation of Curcuma longa leaves illuminated its safety profile, with no observed toxicity at lower doses. However, caution is warranted at higher doses, as evidenced by dose-dependent toxic effects. The LD50 of 2154.06 mg/kg provides a pivotal reference for establishing safe dosage parameters. These findings contribute valuable insights into the safety considerations of Curcuma longa extract, paving the way for informed applications in therapeutic and medicinal contexts. Further studies, including chronic toxicity assessments, will enhance our understanding of the long-term safety implications and facilitate the responsible utilization of this natural remedy.

Acknowledgments

I am thankful to Head of Pinnacle Biomedical Research Institutional, Bhopal, for providing me the facilities to carry out my research work

Funding source: NO 

Competing interests/Conflicts of interest: NO 

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