1. INTRODUCTION:

In Unani Medicine, predisposition to disease and even the selection of a preventive and therapeutic regime is mostly based on a phenotypic evaluation of a person, which includes one's bodily constitution, referred to as "Mizaj". Temperament/Mizaj is a result of the relative proportion of four entities: Dam (blood), Balgham (Phlegm), Safra (yellow bile), and Sauda (Black Bile), which is determined not only by genetics but also by external influences.

In classical Unani writings, distinct traits and functions such as cell division, metabolism, locomotion, intellect, signalling, and so on have been assigned to each of the four entities. This clinical phenotypic variety, according to Unani Philosophy, is the outcome of varying proportions of Dam, Balgham, Safra, and Sauda. The dominance of each of these results in Damwi (Sanguine), Balghami (Phlegmatic), Safrawi (Choleric) and Saudawi (Melancolic) temperaments; the foregoing philosophy serves as the foundation of the Unani System of Medicine¹. Gastritis, often known as hyperacidity, is a prevalent issue among many people. In Unani Medicine, the disorders associated with Meda/stomach are thoroughly documented and understood. Gastritis (mild) or hyperacidity is associated with the Choleric temperament; according to Unani philosophy, Safrawi (Choleric) people are robust and active, with a high metabolic rate and good digestion². Upper abdominal pain, such as heartburn or dyspepsia, is a frequent clinical sign of gastritis. In the modern era, oxidative stress is a significant factor to the onset of gastrointestinal disorders. The stomach is a fragile digestive organ which is vulnerable to a variety of external infections as a consequence of intake of food. Certain microbes, such as Helicobacter pylori, induce oxidative stress in the stomach lining, where it leads to inflammation and may be involved in the development of gastro – intestinal illnesses like gastritis, stomach ulcers, and gastric carcinoma, as well as some systemic diseases like functional dyspepsia³.

Exogenous elements like food and medicine can also produce ROS in the gut ROS like superoxide (O₂−) and hydrogen peroxide (H₂O₂) cause inflammation and tissue damage by fragmenting cellular DNA. The human cytochrome p450 gene CYP3A4 is found on chromosome 7q21.1. The promoter region polymorphism, CYP3A4*2 664 T>C (Ser222Pro) found to be correlated with lower expression and activity (1.7 to 5 times less) and it has been associated as a risk factor for many cancers and oxidative stress-related illnesses ^4,5. Though there have been multiple studies related to CYP3A4*2 (664 T>C) polymorphism to altered drug metabolism, there are very few studies associating such genetic polymorphisms to philosophy of Unani Clinical Phenotyping (as well as Unani Medicine). Glutathione S-Transferases (GSTs) are a class of phase II enzymes that include cytoplasmic, membrane, mitochondrial, and leukotriene C4 synthases. They are implicated in the detoxification of potential carcinogens and provided of a sturdy antioxidant function by neutralizing electrophiles and free radicals with nucleophilic glutathione exuding less toxic products^6,7. As a result, they guard against free radical damage to the cell, protein, and nucleic acid. A reduction in activity might make an individual more vulnerable to several illnesses, including cancers⁸. The GST superfamily's GSTT1 and GSTM1 isoenzymes both have null or deletion polymorphism, leading to absence of enzyme production⁹. Genetic variants are thought to have a major persuade in affecting the varying risks for different illnesses reported within and between populations. Previous investigations on Gastritis, CYP3A4 and GST polymorphisms have yielded equivocal findings¹⁰. Thus, the current investigation aims to discover if a genetically driven decrease in the ability to detoxify electrophilic chemicals, in conjunction with Unani philosophies of disposition, has an essential role in predicting Gastritis risk. This is the first type of research linking the temperament Safrawi (Choleric) to the disease gastritis, and GST genetic polymorphism investigations.

2. MATERIALS AND METHOD:

2.1 Subject selection:

A total of 852 subjects were screened during the reporting period of which 200 subjects were registered for the study, out of which 100 gastritis patients and 100 control subjects (Healthy individuals) of Safrawi (Choleric) temperament were selected based on clinical history and CRF form specially designed by Unani physicians of National Research Institute of Unani Medicine for Skin Disorders (previously CRIUM, Hyderabad). This study was approved by the Institutional Ethics Committee of NRIUMSD, following the principles of Helsenki Declaration. All the gastritis patients were confirmed based on their histopathological reports.

2.2 Blood Sample Collection:

In K2 EDTA vacutainers, two ml of whole blood was obtained by venipuncture from each of the 200 cases (100 patients and 100 controls) and stored in the refrigerator at -4°C until they were needed. Written and informed consent were taken from all the subjects.

2.3 DNA isolation:

HiPurA™ blood genomic DNA Purification Kit (a column-based DNA isolation kit) was used for genomic DNA from peripheral blood samples. To assess the purity and amount of the DNA, Agarose gel electrophoresis and nanodrop reading by using Multimode reader was performed and then stored at -20°C until further use.

2.4 PCR amplification and genotype determination of CYP3A4*2 (664 T>C):

The PCR amplification of CYP3A4*2 664 T>C (rs55785340) was performed¹¹. A total volume of 30μl was used for the PCR reaction, which included 25μl mastermix of 10Xbuffer, dNTPs, forward and reverse primers (Table 1), Taq DNA polymerase, and 5μl 200 g of genomic DNA. In a thermal cycler (Fermentas Life Sciences, Bangalore, India), reactions for the CYP3A4*2 673 TC gene were carried out according to the following programme: 95°C for 2 minutes, 35°C for 1 minute, 59.5°C for 1 minute, 72°C for 1 minute, and 72°C for 5 minutes. To identify correct amplification of the gene of interest segments, PCR products generating a 366-bp fragment were observed on a 2 percent agarose gel stained with ethidium bromide before digestion (Figure 1a). The CYP3A4*2 genotyping was performed by Restriction digestion by HindIII FastDigest restriction enzyme (Thermo Scientific, India). The samples were incubated for 20min at 37°C and the RFLP products were separated by 3% agarose gel electrophoresis (Figure 1b).

(a)

Lane 1,2,3,4 and 5 PCR products 366bp.

Lane L 100bp ladder.

(b)

Lane 1 and 4- TT genotype 180bp

Lane 2 and 3- TC genotype 366bp and 180bp

Lane 5 –CC genotype 366bp

Lane L 100bp ladder

Figure 1: CYP3A4*2 (664 T>C) (rs55785340) gene polymorphism PCR products (a) and RFLP products (b)

2.5 PCR amplification and genotype determination of GSTM1 and GSTT1 genes:

A modified multiplex PCR technique for simultaneous replication of both genes for molecular investigation was used to assess the homozygous and null polymorphism of GSTM1 and GSTT1 ¹²with a globulin gene fragment as an internal positive check for a successful amplification. Primers were given in table 1. The amplified products were examined by 2% gel electrophoresis (Figure 2). With the GSTM1 primers, a 215-bp fragment was amplified by PCR, whereas the GSTT1 primers produced a 480-bp fragment. The null genotypes were consistent in their lack of an amplified product. The PCR settings used to determine GSTM1 and GSTT1 variants were identical to those previously reported

PCR products on gel electrophoresis

L shows 100 bp ladder.

Samples 1,4,7 and 8 shows GSTT1 +,

Samples 3 show GSTM1+,

Samples 5,6and 9 shows GSTT1 + and GSTM1 +.

Figure 2: Typical representation of PCR amplification and Genotyping of GST T1 and GST M1 Gene.

Table1: Primers and PCR products

Gene/SNP Primer

Sequence (5’ to 3’)

Amplicon size(bp)

GST M1 F

GST M1 R

5’-GAACTCCCTGAAAAGCTAAAGC-3’

5’-GTTGGGCTCAAATATACGGTGG-3’

215bp

GST T1 F

GST T1 R

5’-TTCCTTACTGGTCCTCACATCTC-3’

5’-TCACCGGATCATGGCCAGCC-3’.

480 bp

β-globulin F

β-globulin R

5’GCCCTCTGCTAACAAGTCCTAC-3’

5’-GCCCTAAAAAGAAAATCGCCAATC-3’

350 bp

rs55785340

CYP3A4*2 F

CYP3A4*2 R

5’- CCTGTTGCATGCATAGAGG-3’

5’GATGATGGTCACACATATC-3’

366bp(PCR product)

180 and 186 bp(after digestion with HindIII)

Table 2: General characteristics of Subjects and Patients with Choleric Temperament

S. No	Characteristics		Controls (No. of cases)	Gastritis patients (No. of cases)
1.	Male		48	25
	Female		52	75
	Total cases		100	100
2.	Age (Mean±S.D.)		39±11 years	37±11 years
	Age (Range)		20 to 60 years	20 to 60 years
3.	BMI		24.5±4.9	26.6±4.6
4.	Diet	Vegetarian	8	6
		Non-Vegetarian	92	94
5.	Cigarette smoking		16	18
6.	Marijuana users		28	35
7.	Alcohol consumption		15	16

Table 3: Genotypes/Alleles distribution of CYP3A4, GSTT1, GSTM1 gene polymorphisms in gastric patients and controls

Genotypes/Alleles	Patients n (freq.) (n= 100)	Controls, n (freq.) (n = 100)	Odds ratio	CI 95%	P value
CYP3A4
TT	66(0.66)	75(0.75)
TC	22(0.22)	21(0.21)	1.19	(0.601 - 2.358)	0.744
CC	12(0.12)	4(0.04)	3.409	(1.049 – 11.08)	0.061
Allele
T	154(0.77)	171(0.88)
C	46(0.23)	29(0.12)	1.761	(1.054 – 2.942)	0.0401*
GSTM1
Wild (+)	51 (0.51)	94 (0.94)	1
Null (-)	49 (0.49)	06 (0.06)	11.29	(4.96-25.67)	0.00001*
GSTT1
Wild (+)	69 (0.69)	90 (0.90)	1
Null (-)	31 (0.31)	10 (0.10)	4.04	(1.85-8.80)	0.0002*

p<0.05 is considered significant

Table 4: Odds risk estimates of CYP3A4 gene polymorphism in gastritis patients and controls

Model	Genotype	Controls N (%)	Patients N (%)	OR (95% CI)	P-value
Codominant	T/T	75 (75%)	66 (66%)	1	0.091
	T/C	21 (21%)	22 (22%)	1.19 (0.60-2.36)
	C/C	4 (4%)	12 (12%)	3.41 (1.05-11.08)
Dominant	T/T	75 (75%)	66 (66%)	1	0.16
Dominant	T/C-C/C	25 (25%)	34 (34%)	1.55 (0.84-2.85)	0.16
Recessive	T/T-T/C	96 (96%)	88 (88%)	1	0.033*
Recessive	C/C	4 (4%)	12 (12%)	3.27 (1.02-10.52)	0.033*
Over dominant	T/T-C/C	79 (79%)	78 (78%)	1	0.86
Over dominant	T/C	21 (21%)	22 (22%)	1.06 (0.54-2.08)	0.86

p<0.05 is considered significant

Table 5: Combined Genotypes distribution and odds risk estimates of CYP3A4, GSTT1, GSTM1 gene polymorphisms in choleric temperament gastric patients and controls

Genotypes		Controls N (%)	Patients N (%)	Odds ratio	CI 95%	P value
GSTM1 Wild (+)	GSTT1Wild (+)	22 (22%)	83 (83%)	1
GSTM1 Null (-)	GSTT1Null (-)	16 (16%)	03 (3%)	20.12	(5.37-75.29)	0.00001*
GSTT1(+) GSTM1(-)/ GSTT1(-) GSTM1(+)		62 (62%)	14 (14%)	16.71	(7.92-35.24)	0.0001*
GSTT1	CYP3A4
Wild (+)	TT	50	71	1
Wild (+)	TC	14	17	1.169	(0.5284-2.588)	0.8553
Wild (+)	CC	5	2	3.55	(0.6622-19.03)	0.242
Null (-)	TT	16	4	5.68	(1.792-18.01)	0.003*
Null (-)	TC	8	4	2.84	(0.8108-9.947)	0.1667
Null (-)	CC	7	2	4.97	(0.991-24.93)	0.075
GSTM1	CYP3A4
Wild (+)	TT	37	72
Wild (+)	TC	10	19	1.024	(0.4324-2.426)	0.868
Wild (+)	CC	4	3	2.595	(0.5515-12.21)	0.4027
Null (-)	TT	29	3	18.81	(5.373-65.85)	<0.0001*
Null (-)	TC	12	2	11.68	(2.482-54.93)	0.0005*
Null (-)	CC	8	1	15.57	(1.876-129.2)	0.004*

p<0.05 is considered significant

Table 6: Haplotype analysis of CYP3A4, GSTT1, GSTM1 gene polymorphisms in choleric temperament

Haplotype association with response (n=200, crude analysis)
	CYP3A4	GSTT1	GSTM1	Freq	OR (95% CI)	P-value
1	T	+	+	0.5875	1.00
2	T	-	-	0.1175	2.79 (1.57 - 4.96)	0.006*
3	C	+	+	0.1025	0.88 (0.45 - 1.70)	0.7
4	T	+	-	0.075	2.53 (1.15 - 5.56)	0.022*
5	C	-	-	0.0525	12.35(1.71 – 22.69)	<0.0001*
6	T	-	+	0.0325	0.57 (0.19 - 1.71)	0.32
7	C	+	-	0.03	6.38 (0.79 - 51.40)	0.083

Global haplotype association p-value: <0.0001

Table 7: Gene – gene interaction in determining the risk of Gastritis in Choleric temperament

Model	Training Bal. Acc.	Testing Bal. Acc.	CV consistency	P value
GSTM1	0.7050	0.7050	10/10	<0.0001*
CYP3A4,GSTM1	0.7050	0.7050	9/10	<0.001*
GSTT1,CYP3A4,GSTM1	0.7050	0.6993	10/10	<0.0001*

Figure 3: Interaction dendogram for the CYP3A4, GSTT1 and GSTM1 gene polymorphisms in Gastritis susceptibility. The length of the dendogram branch that connects two polymorphisms indicates the strength of interaction (the shorter the branch, the stronger is the interaction).

5. DISCUSSION:

In general, it is thought that reflux of stomach contents is implicated in gastrointestinal illnesses. However, the precise pathophysiological pathways of inflammation and cellular damage during gastro-oesophageal reflux remain unclear. According to current research, oxidative stress is implicated in the pathophysiology of stomach inflammation, ulcerogenesis, and carcinogenesis in H. pylori infection¹⁴. H. pylori, a Gram-negative microaerophilic bacterium, have been demonstrated to cause oxidative stress in infected gastric mucosa, resulting in chronic atrophic gastritis and gastric cancer ⁸. Cytotoxin-associated gene (cage) may trigger gastric epithelial cells' response to oxidative stress and generate reactive oxygen species (ROS) and/or Reactive Nitrogen Species (RNS) often from neutrophils following H. pylori contagion and colonization¹⁵. Polymorphisms in metabolism genes may lead to altered antioxidant defense system in the gastric mucosa, resulting in DNA damage, cell death (including apoptosis), and subsequent cell proliferation, culminating in the development of gastritis and gastric malignancies (figure 4). Antioxidant deficiency has been associated to a variety of additional gastrointestinal disease characteristics¹⁶. Individual habits such as drinking, smoking, and marijuana use were previously thought to play an important role in the development of gastritis by disrupting the balance of antioxidant levels in the gastric mucosa, leading to inflammation^17,18 but the present study found no significant difference in their use between controls and patients. This might be owing to a higher proportion of females and a limited research sample.

Figure 4: A conceptual diagram of the oxidative stress in Gastritis

Cytotoxin-associated gene (cagA) may trigger gastric epithelial cells' response to oxidative stress and generate reactive oxygen species (ROS) and/or reactive nitrogen species (RNS) often from neutrophils following H. pylori infection and colonisation. Polymorphisms in CYP3A4, GSTT1, GSTM1 may lead to altered antioxidant defense system in the gastric mucosa, resulting in DNA damage, cell death (including apoptosis), and subsequent cell proliferation, culminating in the development of gastritis and gastric malignancies.

According to Unani philosophy of medicine, in the human body, there are four senses of humour: Dam (blood), Balgham (Phlegm), Safra (yellow bile), and Sauda (Black Bile) Diseases are caused by their imbalance in the body. In Unani system, gastritis falls under Safrawi (Choleric) a yellow bile altered disease¹⁹. There were no investigations in traditional Unani medicine and genetic diversity studies in oxidative stress /drug metabolism in the relation to gastritis, a Safrawi (Choleric) disease state. Prior studies on gastritis found that the most prevalent cause of gastritis is also due to usage of drugs for various therapies and ingestion of xenobiotic food. Apart from H. pylori infection, improper drug metabolism by CYP enzymes may also result in the formation of highly toxic intermediate molecules, which may trigger the inflammatory process in the stomach^20,21. In this research CYP3A4, GSTT1 and GSTM1 gene polymorphisms and its relation with gastritis in Safrawi (Choleric) temperament were assessed. A prominent enzyme in CYP450 drug metabolizing family, CYP3A4 is an improtant enzyme that contributes to the oxidative metabolism of drugs by producing oxidative intermediates. Thorn et al²² observed mRNA expressions CYP3A4 in all of gastrointestinal mucosa (stomach, duodenum, right and left colon, rectum). Von Richter et al²³ stated that mRNA expression of CYP3A4 in the enterocytes is 3 times higher than in hepatocytes. CYP3A4 enzyme is seen in eradicating H. pylori. It is predicted that the failure of H. pylori annihilation may also be due to gene polymorphisms in CYP3A4²⁴. At the CYP3A4 mRNA level, individual differences and medication interactions alter tissue exposure to reactive intermediates. The CYP3A4 gene's expression and activity varies a lot from person to person. The decreased expression of CYP3A4 mRNA and enzyme activity have been related to the CYP3A4*2 allele^25,26,27. In a recent study related to gastritis and gastric cancer, the Kaplan-Meier analysis demonstrated increased CYP3A4 expression was substantially linked with poorer overall survival and initial progression. As a consequence, CYP3A4 expression may be linked to the potentially carcinogenic transition of chronic atrophic gastritis (CAG) to Gastric Cancer²⁸. In the present study also the mutant alleles of CYP3A4 which were associated with higher expression were shown to be 1.76 folds risky in gastritis patients than in controls.

The conjugation of glutathione by GST has been proposed as a method for intracellular inactivation of alkylating cytostatic xenobiotics. It has been determined that the null allele variant of GSTT1 (22q11.2) increases the risk of gastritis by hindering glutathione's ability to conjugate toxins. GSTM1 is a recycler and glutathione synthase. Its null variant has also been linked to higher the risk of gastric illness and other oxidative stress-related diseases²⁹. According to previous research, both null genotypes had 1.9-2.5 times increased odds of having more serious gastrointestinal illnesses⁹. GSTs expression is generally tissue dependant, and there is a wide variation across humans. GSTM1 is prevalent in the human liver and also other extrahepatic tissues such as the stomach and brain; GSTT1 has been expressed in liver of humans and at very high levels in normal gastric and colonic mucosa³⁰. GSTM1 and GSTT1 genes play an essential role in antioxidant defence mechanisms in gastric tissue; deletion polymorphisms in these genes will exacerbate gastric illnesses, perhaps resulting to deadly diseases such as cancer. Several studies have looked at the association between GSTM1 or GSTT1 null genotypes and their risk for gastrointestinal illnesses, however the findings are assorted ⁷. From earlier analyses, polymorphisms in the GSTT1, GSTM1, and GSTP1 genes are related with an increased risk of gastrointestinal illnesses, particularly malignancies, in the Asian population^31,32,33. Few other studies have also concluded that GST gene polymorphisms are inconsequential in stomach cancer, particularly in the European population³⁴. There aren’t sufficient literatures on the effects of these polymorphisms in gastrointestinal disorders in the Romanian population³⁵. Two investigations in an Iranian and a Chinese population found a link amid the GSTM1 deletion/null variant and gastrointestinal illnesses^36,37. The GSTT1 and GSTM1 null variants were shown to have a substantial correlation with gastritis or hyperacidity in this investigation. It was also evaluated using clinical phenotypes, which were all bilious/Safrawi (Choleric) temperament according to Unani philosophy. GSTT1 and GSTM1 null variants may affect the antioxidant system, resulting to gastritis in Safra/Bilious phenotypes. Considering the significance of both polymorphisms in the defective detoxifying capacity of the stomach, which leads to oxidative stress, further analyses based on the combined GSTM1 and GSTT1 genotype were performed, and it was noticed that subjects with the double null genetic variant and even single null variant were at a significantly higher risk of developing Gastritis than subjects with both genes present. These results are in tune with other studies which are also in agreement with strong association between GSTM1/GSTT1 double null genotype and risk of disease susceptibility in Asian population^38,39,40.

CYP3A4 –TT and GST Null (-); CYP3A4 –TT, TC, CC and GST Null (-) showed significant association with risk of developing gastritis in Safrawi (Choleric) temperament. In haplotype analysis, T-GSTT1 (-) -GSTM1 (-), T-GSTT1 (+)-GSTM1 (-) and C-GSTT1 (-) -GSTM1 (-) haplotypes showed significant association with increased risk towards disease susceptibility, corroborating the hypothesis that these SNPs serve a stimulatory role in illness development in Safrawi (Choleric) temperament. MDR analysis of CYP3A4, GSTT1, GSTM1 gene polymorphisms in choleric temperament showed that the CYP3A4 T>C had an independent effect whereas GSTT1 and GSTM1 have shown a combined effect in gastritis disease susceptibility. Therefore, it is evident that the gastritis is inherited in a multifactorial approach with major/minor gene (s) implication apart from the environmental influences. To recapitulate, all the three gene polymorphisms have a significant role in gastritis susceptibility. According to Unani philosophy, this is the first study to investigate the CYP3A4 and GST polymorphisms in patients with gastritis who have a Bilious/Safrawi (Choleric) temperament.

6. CONCLUSION:

In conclusion, CYP3A4 (TC and CC), GSTT1null and GSTM1null genotypes appear to enhance the risk of gastritis in Safrawi (Choleric) temperament individuals. To reduce illness propensity and reverse pathogenic processes, early diagnosis of these genetic variations is necessary. In fact, proper diet and a healthy lifestyle can help prevent gastritis in its early stages. The strength of our study is the potential assessment of the proposed oxidative stress related genetic variants playing a key role in gastritis in a specific Safrawi (Choleric) temperament. The current study's limitations persist due to its smaller sample size. To exploit personalized therapy approaches, further research examining the genetic diversity in oxidative stress parameters with larger sample numbers in different ethnicities with Safra/Bilious temperament are needed to discover novel susceptibility and prognostic indicators to validate these findings and explore the underlying causes.

7. ACKNOWLEDGMENT:

We acknowledge the financial grant from CCRUM, Ministry of AYUSH, Govt. of India, New Delhi under Fundamental Research Programme and all the subjects who participated in the study.

8. CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

9. REFERENCES:

1. Moalijaat Jild Doom- amraz-e nizam -e-hazam we tawleed we tana sul. Waseem ahmad azmi wazarate terqi insane Wasail Hakumat hind faroog urdu bawan fc-33/9, Institutional Area, Jasooly, New Delhi 110025.page no.207, 2012.

2. Shugufta Nisa. Gastritis (Warm-e-meda): A review with Unani approach. International Journal of Advanced Science and Research 2018; 3(3) 43-45.

3. Suzuki H, Matsuzaki J, Hibi T. Ghrelin and oxidative stress in gastrointestinal tract. J Clin Biochem Nutr. 2011; 48: 122–125.

4. Danielson PB. The cytochrome P450 superfamily: biochemistry, evolution and drug metabolism in humans. Curr Drug Metab 2002; 3: 561–597.

5. Floriano-Sanchez E, Rodriguez NC, Bandala C, Coballase-Urrutia E, Lopez-Cruz J. CYP3A4 expression in breast cancer and its association with risk factors in Mexican women. Asian Pac. J. Cancer Prev. 2014; 15(8): 3805-9. PubMed PMID: 24870798.

6. J. D. Hayes and D. J. Pulford, The glutathione S-transferase supergene family: regulation of GST and the contribution of the isoenzymes to cancer chemoprotection and drug resistance, Critical Reviews in Biochemistry and Molecular Biology, 1995; 30(6): 445–600.

7. M. A. Garc´ıa-Gonz´alez, E. Quintero, L. Bujanda et al., Relevance of GSTM1, GSTT1, and GSTP1 gene polymorphisms to gastric cancer susceptibility and phenotype, Mutagenesis, 2012; 27(6): 771–777.

8. S. Tripathi, U. Ghoshal, B. Mittal, D. Chourasia, S. Kumar, and U. C. Ghoshal, “Association between gastric mucosal glutathione- S-transferase activity, glutathione-S-transferase gene polymorphisms and Helicobacter pylori infection in gastric cancer,” Indian Journal of Gastroenterology, 2011; 30(6): 257–263.

9. Tomomitsu Tahara, Tomoyuki Shibata, Masakatsu Nakamura, Hiromi Yamashita, Daisuke Yoshioka, Masaaki Okubo, Joh Yonemura, Yoshio Kamiya, Takamitsu Ishizuka, Yoshihito Nakagawa, Mitsuo Nagasaka, Masami Iwata, Ichiro Hirata, And Tomiyasu Arisawa. Association between Genetic Polymorphisms Related to DNA Repair or Xenobiotic Pathways and Gastric Premalignant Conditions. Anticancer Res. April 2011; 31(4): 1459-1465.

10. W. Beil, B. Obst, K.-F. Sewing, and S. Wagner, “Helicobacter pylori reduces intracellular glutathione in gastric epithelial cells,” Digestive Diseases and Sciences, 2000; 45(9): 1769– 1773,.

11. G. Suman and Kaiser Jamil. Novel CYP3A4 Gene Polymorphisms in Post Chemo Breast Cancer Patients. International Journal of Cancer Research, 2006; 2: 358-366.

12. Alokananda Chakraborty, Priyanka Pallapolu, Seelamneni Thulasamma, Lahari Kuna, Shravan Kumar Ghali, Kaiser Jamil, Ghazala Javed, Munawwar Husain Kazmi. Genetic variations in NLRP1 and Glutathione S-Transferase are associated with Generalized Vitiligo of Phlegmatic Clinical Phenotypes in Unani Medicine Philosophy of South Indian Population. Research Journal of Pharmacy and Technology. 2022; 15(5): 2221-7.

13. Lou, Xiang-Yang, Chen, Guo-Bo, Yan, Lei, Ma, Jennie Z., Zhu, Jun, Elston, Robert C., Li, Ming D., et al.,. A generalized combinatorial approach for detecting geneby-gene and gene-by-environment interactions with application to nicotine dependence. Am. J. Hum. Genet. 2007; 80(6): 1125–1137 Jun.

14. Qadri Q, Rasool R, Gulzar GM, Naqash S, Shah ZA. H. pylori infection, inflammation and gastric cancer. J Gastrointest Cancer 2014; 45(2): 126–32. doi: 10.1007/s12029-014-9583-1.

15. Butcher, L. D., den Hartog, G., Ernst, P. B., and Crowe, S. E. . Oxidative Stress Resulting from Helicobacter pylori Infection Contributes to Gastric Carcinogenesis. Cellular and molecular gastroenterology and hepatology, 2017; 3(3), 316–322.

16. Suzuki H, Nishizawa T, Tsugawa H, Mogami S, Hibi T. Roles of oxidative stress in stomach disorders. J Clin Biochem Nutr. 2012; 50: 35–39.

17. Bishehsari F, Magno E, Swanson G, Desai V, Voigt RM, Forsyth CB, Keshavarzian A. Alcohol and Gut-Derived Inflammation. Alcohol Res. 2017; 38(2): 163-171. PMID: 28988571; PMCID: PMC5513683.

18. Joundi H, Pereira KN, Haneef G, Bhandari R, Malik J, Shah RP, Sejdiu A, Mathialagan K. Cannabis Use Disorders Lead to Hospitalizations for Peptic Ulcer Disease: Insights from a Nationwide Inpatient Sample Analysis. Cureus. 2021; Jun 2; 13(6): e15405.

19. Zuhr I. "Kitabul Taiseer Fil Madawat", (Translated By CCRUM, New Delhi), pp. 126, 1986.

20. Jasna, D. , Dražen, S. . Oxidative Stress Pathway Driven by Inflammation in Gastric Mucosa. In: Tonino, P., editor. Gastritis and Gastric Cancer - New Insights in Gastroprotection, Diagnosis and Treatments Internet. London: IntechOpen; 2011.

21. M. Vaishali. Antioxidants in Health and Diseases. Research J. Pharm. and Tech. 2014; 7(4): 489-493.

22. Thorn M, Finnstrom N, Lundgren S, Rabe A, Loof L. Cytochromes P450 and MDR1 mRNA expression along the human gastrointestinal tract. Br J Clin Pharmacol. 2003; 60(1): 54-60.

23. Von Richter O, Burk O, Fromm MF, Thon KP, Eiechelbaunm M, Kivisto KT. Cytochrome P450 3A4 and P-glycoprotein expression in human small intestinal enterocytes and hepatocytes: A comparative anallysis in paired tissue specimens. Clin Pharmacol Ther. 2004; 75(3): 172-83.

24. Banet LZ, Kroett DL, Sheinen LD. Pharmacokinetics. In: Hardaman JG, Limbrid LE, Molinoff PB, Ruddon RW, Gilman AG (eds). Goodman and Gilman’s. The Pharmacological Basis of Therapeutics. 9th ed. Mc GrawHill. New York 1996.11-16.

25. M. Sumithra, Avantika Prabhabanik. A Prospective Study of Drug Utilization and Evaluation of Gastro Intestinal Agents. Research J. Pharm. and Tech. 2017; 10(1): 166-170. doi: 10.5958/0974-360X.2017.00036.1

26. Sapone A, Vaira D, Trespidi S, Perna F, Gatta L, Tempieri A, Ricci C, Cantelli-Forti G, Miglioli M, Biagi GL, Paolili M. The clinical role of cytochrome p450 genotypes in Helicobacter pylori management. Am J Gastroenterol. 2003; 98(5): 1010- 15.

27. Koch I, Weil R, Wolbold R, Brockmöller J, Hustert E, Burk O, Nuessler A, Neuhaus P, Eichelbaum M, Zanger U, Wojnowski L. Interindividual variability and tissue-specificity in the expression of cytochrome P450 3A mRNA. Drug Metab Dispos. 2002; Oct; 30(10): 1108-14.

28. Zhang F, Wang F, Chen C, Wang T, Hu J, Su R, Li X, Gu B, Tang S, Chen H, Li Y. Prediction of progression of chronic atrophic gastritis with Helicobacter pylori and poor prognosis of gastric cancer by CYP3A4. J Gastroenterol Hepatol. 2020; Mar; 35(3): 425-432. doi: 10.1111/jgh.14844. Epub 2019 Sep 12. PMID: 31441961.

29. Lao X, Peng, Q, Lu, Y, Li S, Qin X, Chen Z, Chen J. Glutathione S-transferase gene GSTM1, gene-gene interaction, and gastric cancer susceptibility: Evidence from an updated meta-analysis. Cancer Cell Int. 2014; 14: 127.

30. Tulsi Rani Thakre, Abha Singh, Mitashree Mitra. Investigation on Glutathione-S-transferase M1 and T1 gene polymorphisms as risk factor in Cervical Cancer. Research J. Pharm. and Tech. 2016; 9(12):2295-2300. doi: 10.5958/0974-360X.2016.00462.5.

31. S. Ghatak, R. P. Yadav, F. Lalrohlui et al. Xenobiotic Pathway Gene Polymorphisms Associated with Gastric Cancer in High-Risk Mizo-Mongoloid Population, Northeast India. Helicobacter, 2016; 21(6): 523–535,.

32. Sergey Kotelevets, Sergey Chekh, Zukhra Karakotova. Modern Possibilities of the Use of Non-Invasive Serological Biomarkers Severe in Population Studies. Research J. Pharm. and Tech. 2019; 12(9): 4274-4282. doi: 10.5958/0974-360X.2019.00735.2

33. Deepalakshmi M, Ajay Samraj P, Diya C, Jonna Venkatesh, Kamalrathinam R S, Arun K P. Case Report on Mild Anemia and Gastritis due to Zidovudine, Lamivudine and Nevirapine (ZLN) Regimen. Research Journal of Pharmacy and Technology. 2021; 14(11): 5911-2. doi: 10.52711/0974-360X.2021.01027

34. Agudo A. Sala N. Pera G. et al. Polymorphisms in metabolic genes related to tobacco smoke and the risk of gastric cancer in the European prospective investigation into cancer and nutrition Cancer Epidemiol. Biomarkers Prev. 2006; 15: 2427–2434.

35. Shiv Kumar Patel, Moumita Sinha, Mitashree Mitra. Glutathione-S-transferase M1 and T1 gene Polymorphism as Risk Factors of Oral Squamous Cell Carcinoma: A Preliminary Investigation. Research J. Pharm. and Tech. 2012; 5(7): 918-920.

36. Cai L, Yu SZ, Zhang ZF. Glutathione S-transferases M1, T1 genotypes and the risk of gastric cancer: a case-control study. World J Gastroenterol 2001; 7:506–9.

37. Cai L, Yu SZ, Zhang ZF. Glutathione S-transferases M1, T1 genotypes and the risk of gastric cancer: a case-control study. World J Gastroenterol. 2001; 7: 506–9.

38. Sukhpal Singh, Amita Mahajan, Jaspreet Kaur. Studies on Status of Oxidative Stress related Molecules and Enzymes in Obese with and without Diabetes in the Northern region of India. Research J. Pharm. and Tech. 2020; 13(2): 801-809. doi: 10.5958/0974-360X.2020.00151.1

39. Dunna NR, Vure S, Sailaja K, Surekha D, Raghunadharao D, Rajappa S, et al. Deletion of GSTM1 and T1 Genes as a Risk Factor for Development of Acute Leukemia. Asian Pac J Cancer Prev. 2013; 14: 2221–4. doi: 10.7314/apjcp. 2013.14.4.2221).

40. Preety Sweta Hembrom, Jisna Jose, Shalini K, Tony Grace. Detection of Variation in Expression of Insecticide Resistance Gene Cyp4d2 involved in Detoxification of Insecticides in Drosophila melanogaster. Research J. Pharm. and Tech. 2019; 12(11): 5315-5319. doi: 10.5958/0974-360X.2019.00921.1

41. Huda Kadhem Mohseen, Baydaa Taher Sih, Sura Salah Al-Saidi. Effect of the (ɣ-ray) and Laser Radiation on the Important Antioxidant Enzyme Glutathione(GSH) level in Serum. Research J. Pharm. and Tech. 2017; 10(10): 3386-3390. doi: 10.5958/0974-360X.2017.00602.3.

Received on 11.09.2023 Modified on 16.02.2024

Research J. Pharm. and Tech 2024; 17(6):2804-2812.

DOI: 10.52711/0974-360X.2024.00440

(b)

Lane 1 and 4- TT genotype 180bp

Lane 2 and 3- TC genotype 366bp and 180bp

Lane 5 –CC genotype 366bp

Lane L 100bp ladder

Figure 1: CYP3A4*2 (664 T>C) (rs55785340) gene polymorphism PCR products (a) and RFLP products (b)

PCR products on gel electrophoresis

L shows 100 bp ladder.

Samples 1,4,7 and 8 shows GSTT1 +,

Samples 3 show GSTM1+,

Samples 5,6and 9 shows GSTT1 + and GSTM1 +.

3. STATISTICAL ANALYSIS: