INTRODUCTION:

Hypothyroidism is a hypo metabolic clinical state resulting from inadequate production of thyroid hormones for prolonged periods, or rarely, from resistance of the peripheral tissues to the effects of thyroid hormones¹.

Alpinia galangal (L) Willd.(Family- Zingiberaceae), a perennial, aromatic, rhizomatous herb used in both traditional as well as in modern system of medicine to treat various physiological conditions. It is an important source of various types of compounds with diverse chemical structures as well as pharmacological activities^2-3.

Chemical constituents:

The earlier studies reveals the presence of various phytochemicals in the rhizomes of Alpinia galangal, which includes terpenoidal moieties, the pungent principal compounds such as 1’S’-1’-acetoxychavicol acetate^4-6. The rhizome also contains flavonoids, some of which have been identified as kaemperol, kaempferide, galangin and alpinin. Kaempferide, galangin and alpinin_.The rhizome of Alpinia galanga posses Anti-inflammatory, analgesic, hypoglycemic, hypolipidemic, anti-allergic, anti-microbial and anti-platelet activity^7-13.As the plant is rich in various polyphenols and exhibits different pharmacological properties, which encouraged us to investigate Alpinia galangal rhizome for its efficacy in management of hypothyroidism.

MATERIAL AND METHODS:

Drugs and chemicals:

Propylthiouracil (PTU) and L-thyroxine were purchased from Sigma- Aldrich. All the solvents and other chemicals used were analytical grade.

Experimental animals:

Forty two female Wistar rats (weighing approximately 150-200g) were procured from animal house of the Institute, maintained under standard conditions in the animal house room at 25.0^oC± 2.0^oC, Relative Humidity 55.0% ± 5.0% with 12h light: 12h dark cycles with a standard pellet diet and water ad libitum. The animal handling and experimental procedures were performed in accordance to the Committee for the purpose of control and supervision of Experiments on Animals (CPCSEA) guidelines and Institutional Animal Ethical Committee (IAEC), Siddhatha Institute of Pharmacy, Dehradun, Uttarakhand (India) approved experimental protocol (Ref. - SIP/IAEC/PCOL/08/2019).

Collection and Authentication of Plant material:

Rhizome part of Alpinia galanga was obtained from a commercial supplier and authenticated by Dr. Sunita Garg, Emeritus Scientist, RHMD (Raw material Herbarium and Museum), CSIR-NISCAIR, New Delhi. A voucher specimen has been deposited at the NISCAIR Herbarium (Ref no. NISCAIR/RHMD/Consult/2019/3524-25-2).

Extraction of plant material:

Preparation of Extracts:

Fresh Rhizome of Alpinia galanga were shade dried and powdered coarsely in a grinder. The powder plant material (500g) was extracted by hot percolation method using soxhlet apparatus with Petroleum ether (make -Thermo Fischer Scientific). The marc of Pet. Ether extraction was further successively extracted by methanol followed by water using cold maceration technique for 72 hrs with regular stirring. After 72 h, the extract was filtered and fresh solvent was added and stirred for next 72 hrs. This procedure was repeated for 2 more times with the fresh solvent. Afterwards the extract was filtered and concentrated under reduced pressure. The concentrated extract was stored at 4^oC for further examinations.

Phytochemical Screening:

Preliminary phytochemical screening of the extracts was done to test the presence of active constituents such as carbohydrates, glycosides, alkaloids, flavonoids, tannins, terpenoids, saponins.¹³

Acute Oral toxicity Study:

An acute oral toxicity study was carried out according to OECD guideline 425. Six Female Wistar strain rats weighing 150-200gm, were administered 2000mg/kg of test solution. After administration of test solution the rats were observed for behavioral changes and mortality at the interval of 0.5 h, I h, 2 h, 4 h, 24 h, 48 h and 72 h and lastly upto 14 days.

Induction of Hypothyroidism:

Hypothyroidism was induced by oral dose of PTU (60µg/kg body weight) in drinking water of Female Wistar Rats for one month. After one month hypothyroidism was confirmed by elevated serum TSH level.¹⁴

Experimental design:

Female wistar rats were divided into 6 groups containing 6 animals each. Group I (normal control) were fed with standard diet and water and were not given any treatment; Group II, III, IV, V and VI receives propylthiouracil (PTU) at a dose of 60µg/kg body weight orally daily upto the induction of disease. Group II serves as disease control and receives only PTU. Group III serves as Standard control and receives PTU and standard drug Levothyroxine (10µg/kg) orally. Group IV, V and VI serves as test groups and receives PTU and test extracts orally with doses of 200mg/kg of each extract respectively.

The body weight and temperature of Rats of all the groups were measured at an interval of 1 week and at the end of 1 month thyroid function test was performed. Total duration of research protocol was two months. At the end of second month, animals were subjected to high dose of thiopental sodium, and then blood was collected from retro-orbital plexus and serum was separated by centrifugation at 2500rpm for 10min. Biochemical parameters of the separated serum were assayed in pathological laboratory.

Histopathological studies:

Thyroid tissue isolated from all experimental groups were fixed in 20% formalin and embedded in paraffin wax. Microtome section of 5-6 micron thickness were cut and stained with haematoxylineosin (H&E) dye. These sections were examined under the Brightfield microscope, for altered histopathological changes in test and control group.

Statistical analysis:

Results are reported as Mean± SEM. The analysis was performed using Graph pad prism version 7 with one-way analysis of variance (ANOVA) followed by Tukey’s test. P value <0.05 were considered statistically significant.

RESULTS:

Phytochemical screening:

Preliminary phytochemical screening shows that the methanolic extract contains flavonoids, triterpenoids, carbohydrates, alkaloids, phenolics and tannins.

Acute oral toxicity study:

Extracts of Alpinia galanga did not show any sign of toxicity or mortality at a dose level of 2000 mg/kg. Hence 1/10^th (200mg/kg) of the drug was selected.

Effect on serum T3, T4 and TSH levels:

There was a significant increase in serum TSH levels in PTU induced wistar rats which confirms the presence of Hypothyroidism. Further it was observed that methanolic extract significantly decrease the levels of TSH (P<0.05) and increase T3 and T4 (P<0.05, P<0.01).

Group	Treatment	T3	T4	TSH
Group	Treatment	nmol/L	nmol/L	mE/L
I	Normal Control	0.75 ± 0.010	47.78 ± 0.523	1.53 ± 0.136
II	Disease Control	0.51 ± 0.013	22.27 ± 0.580	9.51 ± 0.186
III	Standard Control	0.73 ± 0.012⃰	43.65 ± 0.576⃰⃰	1.43 ± 0.0147⃰ ⃰
IV	Pet. Ether ext.	0.73 ± 0.010⃰	42.72 ± 0.359⃰	1.89 ± 0.044⃰
V	Met. Ext.	0.73 ± 0.009⃰	43.97 ± 0.681⃰ ⃰	1.82 ± 0.101⃰ ⃰
VI	Aq. Ext.	0.72 ± 0.020⃰	42.82 ± 0.443⃰	1.85 ± 0.161⃰