Survey of Snuff Use and Preliminary Study of Effect of Two Brands on, Brain Antioxidants and Acetylcholinestrase Enzyme of Wister Albino Rats

INTRODUCTION

Snuff is any product made from ground or pulverized tobacco leaves intended to be placed in the oral or nasal cavity.¹ This distributes rapid nicotine sensation with long-lasting fragrance and essence. Snuff could be placed between lips and gum (moist snuff), or sniffed through the nose (dried powdered tobacco product). For instance “Naswar” a dipping smokeless tobacco product commonly used in Pakistan, Afghanistan, Iran and South Africa.² Snuff became common in the United Kingdom throughout the seventeenth century, but powdered tobacco is documented to have been used by native inhabitants of Brazil before the coming of the Spaniards.³ In the western world, Snuff reemerged as substitutes, for tobacco and became generally acceptable among younger generations after the ban on smoking in many public places. The traditional snuff production involves the selection of varieties of tobacco leaves, sun-dried then subjected to fermentation process which gives a characteristic scent. It is converted to powder known as snuff blend which is mixed with calcium oxide and wood ash (original fine snuff without addition of flavors). However, most often varieties of spice, piquant, fruit, flora and menthol are added to the pure or blend snuff. The types of snuff include dry, wet, dipping tobacco, chewing tobacco, and creaming snuff.⁴

Another form of snuff made from moringa (drumstick) are now widely accepted and sold in some African countries including Nigeria. Although they are a blend of chiefly moringa leaves, they are not devoid of tobacco. Moringa snuffs are affectionately consumed by car drivers, motorcyclists, menial laborers and even some members of the elite. They are sold under brand names such as : Special moringa sundu, AK-47 boss, Hajiyah aisha man power, Sweet mother, Normal tobacco, lion brand, Hajiyah Bilkisu, Hajiyah Fatima, Hajiyah Bilkisu Ma`a shaa Allah, Shehu Barhama, and Hajiyah Hauwa.

Recent studies suggested that long term use of smokeless tobacco could predispose to free radical generation and oxidative stress.^{5, 6} Nicotine is a main bioactive component of tobacco. Previous studies have shown that low doses of nicotine can improve memory function and reduced plaque burden and could be used as anti-Alzheimer disease agent and improved attention performance in schizophrenia patients.^{7, 8} However, some studies have indicated that nicotine impairs cognition in human and animal subjects. These inconsistencies could be attributed to the doses. It appears high dosage of nicotine might induce neurotoxicity. ⁹ and excite oxidative stress, while low amount could improve cognitive performance.⁸ On the other hand, the major component of the modern West African snuff is moringa oleifera a plant considered as one of the most beneficial trees in the world, with several medicinal, nutritional and industrial applications. ^{10, 11} Moringa is rich in vitamins, antioxidants, β-carotene, amino acids, phenolics, and flavonoids.¹² These various components of moringa make potent free radical scavengers, enzyme inhibitors, antioxidants, anti-bacterial, anti-tumor, cholesterol lowering, antipyretic, anti-inflammatory, anti-diabetics’ anti-ulcer among others. ¹³

The rate of moringa snuff consumption has rampantly increased in Nigeria particularly the Northern region; where in the past, snuffing was considered to be filthy. The addiction to snuff is obvious, although the addicts claim that, it has various therapeutic benefits against different ailments.  Unlike the “traditional snuff” (i.e. a blend of purely tobacco and flavouring agent) whose effect has been studied and documented to cause disorders such as cancer (of the mouth, lips, nasal cavities, oesophagus and gut), diabetes; hypercholesterolemia, myocardial infarction and  teratogenicity,⁶ modern snuff contains high percentage of moringa beside other adjuncts and was never studied. For this reason, the current study aimed to survey some brands of snuff and the effect of special moringa sundu (SMS) and smokeless tobacco (ST) on brain antioxidants and acetylcholinestrase (AchE) in vivo.

MATERIAL AND METHODS

Chemicals and kits

The chemicals used in this research include disodium hydrogen phosphate (Na₂HPO₄), sodium dihydrogen phosphate (NaH₂PO₄) sodium chloride (NaCl) and ethanol 95% and isoflurane were purchased from Fluka, Switzerland. All kits used where obtained from Solarbios Life Science Limited, Beijing China. These include Superoxide dismutase (SOD) Catalog Number: BC0175, Glutathione Peroxidase (GPX) Catalog Number: BC0174, Malondialdehyde (MDA) Catalog Number: BC002, reduced glutathione (GSH) Catalog Number: BC1175), Acetylcholinesterase (AchE) Assay Kit Catalog Number: BC2020

  Snuff samples, composition and preparation

The products figure 1 (smokeless tobacco and special moringa sundu) were purchased from retail shop in keffi town, Nasarawa State, Nigeria. Based on manufacturer’s description, Smokeless tobacco contains ground tobacco leaves and menthol while special moringa sundu contains tobacco powder, menthol and moringa powder.

To prepare snuff solution; 1g of sample was dissolved in 1 liter of deionized water and refrigerated until used.

Fig. 1:  Snuff samples: A. Special Moringa Sundu; B. Smokeless Tobacco

A: Commercial product containing a mixture of moringa powder, tobacco powder and menthol

B: Commercial product containing finely pulverized dried tobacco leaves with neither sweetener nor flavourings

Experimental Design:

The study was carried out in two phases. The first phase involved the use of questionnaire to obtain relevant information from snuff users. This provided an insight into the average quantity of each snuff consumed and also the reward-seeking behavior of the users.

The second phase i.e. the in vivo study in rats which was approved by the Nasarawa State University Keffi Animal Ethics Committee, involved the use of smokeless tobacco, special moringa sundu, and thirty (30) Wister albino rats (weighing 110-120g). The snuffs were obtained from shop number nine (9) Kaduna road Angwan NTA keffi, Nasarawa state; the antioxidant and acetyl cholinesterase enzyme kit used were obtained from Solarbios Life Science Limited, Beijing China while the rats were obtained from National veterinary research institute (NVRI) VOM, Plateau state.

These rats were housed in cleaned plastic cages, bedded with clean rice husks, a 12 hour light: 12 hour dark cycle and free access to food and water ad libitum. After two (2) weeks acclimation period, the animals were weighed and randomized into five (5) designated groups of six (6) rats each: Group 1 (control received no treatment ), Group 2 (received a dose of 6mg/Kg body weight smokeless tobacco), Group3 (received a dose of 3mg/Kg body weight smokeless tobacco), Group4 (received a dose of 6mg/Kg body weight special moringa sundu), and Group5  (received a dose of 3mg/Kg body weight special moringa sundu)

All animals received humane treatment; they were fed with growers mesh and allowed access to water throughout the treatment period (two months). Food gavage was used in the administration of the solution.

 At the end of the experiment (administering of snuff solution) rats were weighed, sedated using 5% V/v isoflurane and  oxygen 1.5L / min flow, the skulls were dissected quickly and the brain tissues were quickly removed and washed in phosphate buffer solution of pH 7.4. The brain tissues were instantly placed in ice and refrigerated at -20°C for 24hrs.

Preparation of Brain homogenate

Brain tissues were cut into pieces, weighed and two (2g) grams was homogenized in 20 volumes of phosphate buffer saline  pH 7.4 (0.1M), at 700g in ice for 10min, then centrifuged for 15min at 5000 rpm . The supernatant was then collected and stored at -20 °C until use. ¹⁴

Biochemical analysis

Determination of superoxide dismutase (SOD) Activity

SOD activities were determined colorimetrically according to manufacturer’s (Solarbios Life Science) instructions. SOD catalyzes the conversion of superoxide molecules (O₂^-) into either molecular oxygen (O₂) or hydrogen peroxide (H₂O₂). The O₂^- reduces nitro-blue tetrazolium to form blue formazan, which absorbs at 560 nm. SOD reacts with O₂^- and suppresses the blue reaction. The intensity of the blue color is inversely proportional to the SOD activity.¹⁵

Glutathione Peroxidase (GPx) Activity  

The Glutathione Peroxidase Activity Assay kit was used to measure glutathione peroxidase in the tissue extracts according to manufacturer’s protocol. The assay is based on the oxidation of reduced glutathione (GSH) to oxidized glutathione (GSSG) which is catalyzed by GPx In the presence of GSH, glutathione reductase and NADPH.  Glutathione peroxidase activity corresponds to oxidation of NADPH to NADP. Absorbance was measured at 340 nm.¹⁶

Determination of total reduced glutathione (GSH)

Glutathione (GSH) is a natural three peptide containing sulfhydryl (SH). It is composed of glutamic acid, cysteine and glycine; it  Glutathione react with 5,5’-dithiobis-2-nitrobenoic acid (DTNB) to give a yellow product that absorbs maximum ally at 412nm. The amount of GSH was determined as GSH (μg /g tissue). ¹⁷

Determination of Malondialdehyde (MDA) level

Malondialdehyde (MDA) is one of the convenient markers of lipid peroxidation. At an acidic pH and high temperature, MDA and thiobarbituric acid (TBA) condenses to form  brown red 3,5,5- three methyl sulfamethoxazole -2,4-two ketone  with  largest absorption wavelength of  532 nm. The level of MDA was calculated by the difference between the absorbance at 532 nm, 450 nm and 600 nm as instructed by the kit’s manufacturer.¹⁸  

Determination of acetylcholinesterase (AchE) Activity

AchE catalyzes Ach hydrolysis to generate choline, and choline reacts with 5,5′-dithiobis(2-nitrobenzoic acid) to form 5-mercapto nitrobenzoic acid (TNB) which absorbs at at 412 nm. AchE activity was expressed as U/g tissue as described by the kit manufacturers.¹⁹

RESULTS

The result in table1 revealed the response of 300 active snuff users. 70 % use combination of different brands for sexual enhancement while 40 % use Special moringa sundu (SMS) for sexual enhancement. 45 % claimed SMS relieves headache, improves vision, and relieves itching, while 15 % claimed Smokeless tobacco (ST) relieves eye itching, fever and tooth ache. SMS comes in 16g container while ST comes in 14g container. From the data obtained, 75 % of users finish a container in two weeks, 15 % in three weeks and 10 % in four weeks irrespective of the brand.

LD₅₀ Determination of different snuff products.

The results of the acute toxicity studies of various snuff products showed mild toxicity, such as difficulty in breathing, shivering, tremors, anxiety and aggressive behavior. The LD₅₀ was estimated to be 2750 mg / kg body weight and 2800 mg/kg body weight for special moringa sundu and smokeless tobacco respectively.

The result revealed a non-significant (P˃0.05) decrease in SOD and GPx activities in all treatment groups with concomitant increase in GSH levels when compared to the control. On the contrary, a significant (P˂0.05) increase in MDA level was observed in groups 3, 4 and 5 while group 2 showed a decrease. Furthermore, the AchE activity decreased significantly (P˂0.05) in both groups 2 and 3 but increased in group 4 and 5 significantly, when compared to the control.

Table 1: Survey of some Snuff Brands commonly consumed in Nigeria. The results in table1 revealed about 300 people were interviewed through a questionnaire, on the consumption rate/doses, effects experiences and the two brands consumed. 

Table 2: Effect of Snuff on Brain Levels of SOD, GPx, GSH, MDA and AchE Activity of Wister Albino Rats.

Results are expressed as Mean ± SD, n=6 values in each column having different superscript (a, b, c, d, e) are significantly different at p˂0.05. GP = Group, ST = smokeless tobacco, SMS = special moringa sundu.