Ayurveda Treatment (Virechana and Basti) and Changes of Intestinal Microbiota at Phyla and Species Level View PDF

Ayurvedic therapies and medical practices have been elaborated for some patients in Japan. Ayurveda means the science of life in Sanskrit, emphasizes the adoption of many healing therapies, which can purify and rejuvenate the body, mind, and soul [1-4]. The medicinal form of science is not just a healing system but also an art of appropriate, healthy, and disease-free living [5-7].

Ayurvedic medicine’s central concept is the theory that health exists when there is a balance between the three fundamental bodily bio elements, called Dosha, which is composed of Vata, Pitta, and Kapha. Doshas are the forces that create the physical body. They determine conditions of growth, aging, health, and disease. Typically, one or two of the three Doshas predominates and picks a constitution or mindbody type. By understanding individual habits, emotional responses, and body type, practitioners can adapt their practice accordingly.

A traditional Ayurvedic detoxification program referred to as Panchakarma (consisting of five varieties of purificatory therapy) aims to help the body re-establish a healthy metabolic system and immunity [8].

The characteristic of Ayurvedic treatment is detoxification with a large amount of oil treatment by body surface oil massage and purgation therapy (or eliminating therapy) with ghee or specially arranged herbal oil [9]. Virechana therapy is an effective Ayurvedic treatment that can cure many health problems naturally. The Virechana therapy detox program, which may take seven to 14 days, is reported safe from any side effects.

Ayurveda treatments focused on alleviating any excesses Dosha (illness) via powerful herbs and/or through the improvement of general lifestyle practices such as Dinacharya (daily Ayurvedic rituals practiced regularly, help to support a life of optimal wellness through routine), Pranayama (the regulation of the breath through certain techniques and exercises) and Meditation [3].

Panchakarma therapy encompasses five treatments that can prevent and heal a number of illnesses. Virechana therapy is defined as the medicated purgation therapy, which cleanses the excess Pitta, leading to purifying blood by clearing the toxins from the body [10]. The treatment concentrates on the lipophilic toxins that are accumulated in the liver and gall bladder. The gastro-intestinal tract is also cleansed by Virechana therapy. Virechana, a purification therapy, causes severe diarrhea, but the effects on intestinal microbiota have not been well studied yet. In this study clinical products and changes of intestinal microbiota during 11 days of Virechana therapy and 5 or 6 days change by Basti Therapy (alteration of decoction and oil enema) are reported.

Subjects

Participants in the current Ayurveda therapy camp were recruited from patients who had been treated in the Hatai Clinic in Tokyo. They were nine females and four males, with various complaints (Table 1). They are requested to fulfill the questionnaire that was used in the GENKI study [11,12]. It focuses on dietary habits, health status, current illness, family condition, and food preference. Food intake can be calculated from a semi-quantified food frequency questionnaire. Body composition, such as water, muscle, and fat%, was measured during the Tanita Impedance Equipment [13].

Two doctors and the several staffs were accompanied to support this program. Dr. Shiho Oikawa in Panchakarma, who was the registered Ayurveda doctor, graduated from Gujarat Ayurveda University India.

The Ethical Committee approved the study design of the Life Science Promoting Association (No. 2018-3).

Treatment

Three treatment methods were applied to the participants according to their condition [2-4]. Four patients received a full course of Virechana therapy for 11 days, and two received Sastikashali pinda sweda (milk porridge Sudation therapy), and one received daily oil. Basti and remedy were done for 11 days. Other six people received Swedana Basti therapy, oil massage, and treatment for 5 or 6 days. Milk porridge sudation therapy is meant to improve general strength, including muscle tone, flexibility of the joint movements, and motor impairment syndrome [14].

Two doctors were participated in the program as controls without receiving Ayurvedic therapy (Table 1).

Table 1: Outline of participants in the Ayurveda treatment camp at Kawaguchi Lake.

In Virechana therapy, patients drank ghee for four days subsequently whole-body oil massage for three days as the pretreatment, then drank 50ml castor oil on the 9th day. It induces multiple purgations on the day, so the diet needed to change to thin rice porridge and drank two litter raisins juice on the same day to avoid dehydration. After the purgation completed from the next day onwards, they are recommended to take a soft meal avoiding wheat and bread easy to digest.

The small piece of feces was collected four times in the sample tubes at the time of admission, after castor oil drinking, just before discharge (11thday), and then after three weeks at home. The treatment schedule is shown below (Figure 1).

Virechana therapy (upper figure): Day 1. Swedana: Sweat subjects’ whole body in a steam box to improve metabolism and dry their body. Day 2-5 Snephapana (internal oleation): After excretion in the morning, face washing and brushing, take 30ml of ghee warmed to human skin temperature at 5:30 am. Then eat nothing until to be hungry. If feel hungry around noon, so eat about half of the usual meal. 

They have consumed oil inside the body to soften the damaged Dosha. On days 2-5, repeat the same procedure as the previous day with a daily increase of ghee from 60 ml to 120 ml. Day 6-8 Abhyanga and Swedana: Perform oil massage on the whole body, including the head and sweat from the entire body in the steam box. Infiltrate oil quality from the body surface. On Day 9 purgation was done: Drink laxatives prescribed at 9:30 am (no breakfast). To excrete Doshas collected in the digestive tract. Till the end of purgation, the patient drank two litter raisins juice little by little. Thin rice gruel could be taken at night; Day 10, 11 Samsarjana Krama: Half rice porridge, 70 % rice porridge, and gradually return to the normal diet. After that, patients returned to a normal diet by 5 to 7 days.

Basti therapy (lower figure): Day 1: Matra Basti: After a wholebody oil massage, including the head, steam sweating was performed, and then 50 to 100 ml of medicated oil was administered from the anus. Keep it until the next day of excretion. On Day 2 to 5: Niruha Basti and Matra Basti: Niruha Basti is performed in the morning empty stomach. After massage and sweating as in the previous day, a 300-750 ml decoction-based drug was administered from theanus. Excretion desire occurs in 5 to 20 minutes and evacuates the bowel contents to the toilet. After Lunch can be taken after excretion. Matra Basti is performed in the afternoon to apply oil massage to the abdomen, waist, thighs, and Swedana with a hot towel or steam, and administer 50 to 100 ml of medicinal oil from the anus, which was done after Lunch.

Day 6. (Last day) Matra Basti: After a light breakfast, whole body oil massage, steam sweat was performed, and 50 to 100 ml of medicinal oil was administered from the anus. Discharge in the afternoon when bowel movement was settled.

The amount of ghee to drink, the number of days, the combination of herbal medicines taken, the menu of meals, etc. was changed depending on the patient’s physical condition and physical strength. The drugs used for the purgation therapy vitiate the Doshas and bring them into the abdomen. While performing the Virechana therapy, the vitiated Doshas are eliminated through the rectum.

We have arranged in Japan to split the decoction enteral in the morning and the oil enteral in the afternoon and do both types of Basti on the same day as we did on the second to fourth day. In India, only a kind of enteral is done per day.

Dietary Therapy

The menu of meals was performed in Japanese style food by the Ayurveda dietitian. During the procedure, the patient is subjected to Oleation first, then Fomentation, followed by Purgative and Samsarjana Karma (post-operative therapy). The internal Oleation is followed for three to seven days. After that, a medicated steam bath is performed for three days. A light and warm diet is prescribed for the patient a day before starting Virechana therapy. However, certain factors like body and mind constitution, age of the person, a mental condition should be considered while opting for Virechana therapy karma.

Main meals: Rice with cereals (500 g of rice), clear soup (Leek onion, winter melon, ragged kelp, rock salt, soy sauce), Sauce of Tsurumurasaki (vine, rock salt, black pepper, unroasted sesame oil), Purple radish with kalonji (purple radish, rock salt, kalonji, Taihaku sesame oil), Tempura of lotus root and carrot (lotus root, carrot, rock salt, millet, soy sauce, sesame, unroasted sesame oil), Cumin rice (rice, barley cumin, ginger, ghee), Eggplant and beans curry (onion, cherry tomato, eggplant, beans, unroasted sesame oil, cumin, mustard seed, turmeric, rock salt), Bean curry with winter melon (winter melon, mung bean, ginger, ghee, cumin, turmeric), Okra spice sauté (okra, turmeric, coriander seeds, rock salt, unroasted sesame oil), Boiled dried figs with cinnamon (dried figs, cinnamon), Buttermilk (yogurt, rocksalt, cumin, ginger, mustard seed, fenugreek,turmeric, ghee) Milk tea with spices (cinnamon, cardamom, clove, tea, milk, cane sugar), Ginger rice (rice, barley, ginger, kelp, rock salt, Shiso leaves, sesame), Corn soup (corn, onion, ghee, rock salt), sautéed radish with sauce (daikon, sesame oil, kuzu, Mitsuba leaf, ginger), soak spinach and ginseng (spinach, ginseng, ginger, kelp stock, rock salt, roasted sesame oil, sesame), Punch holon saute of yam (yam, mustard seed, cumin, fennel, karonji, fenugreek, were served during the camp (Figure 2). Black pepper, cumin, brown mustard, coriander, turmeric, garam masala, cinnamon, cardamom, clove, kalonji, ajowan were used for spice [14].

Various kinds of oils, such as ghee, olive oil, white sesame oil, roasted sesame oil, perilla oil, and coconut oil, were frequently used. Rice porridge and mung beans are a digestive meal that helps to recover weak internal organs.

Daily energy intake was adjusted between 1200-1500 kcal (about 30 kcal/kg body weight) depending on the patients’ body weight.

DNA Extraction from Fecal Samples

DNA extraction and 16S rRNA was performed as previously described (Vet Microbiol 2020 Uchiyama). Briefly, the genomic DNA was extracted from the samples using Chemagic DNA Stool 200 Kit (PerkinElmer, Waltham, MA, USA). The V3-V4 regions of the 16S rRNA gene were amplified by PCR and subjected to pair-end sequencing using Illumina MiSeq. The sequence data were processed using Quantitative Insights into Microbial Ecology 2 (QIIME 2) v2019.4.0. The DADA2 software package v2019.4.0 incorporated in QIIME 2 was used to correct the amplicon sequence errors and construct an amplicon sequence variant (ASV) table. The Green genes 99% reference database v13.8 was used for the taxonomic classification of each ASV. Microbial taxonomy was assigned using a Naïve Bayes classifier trained on the SILVA 138 database. The sequence data were processed using Quantitative Insights into Microbial Ecology 2 (QIIME 2) v2019.4.0. The DADA2 software package v2019.4.0 incorporated in QIIME 2 was used to correct the amplicon sequence errors and to construct an amplicon sequence variant (ASV) table. More details are described in the previous paper [15-18].

The fecal microbiota of 15 subjects was analyzed using the 16S rRNA amplicon sequencing method. A total of 2,032,860 nonchimeric reads (39,860.0±7,307.8 nonchimeric reads/sample; mean±SD) were used in this study. To rarefy the data, we used 24,000 reads from each sample.

Statistics

Microbial taxonomy was assigned using a Naïve Bayes classifier trained on the SILVA 13299 % database. First, changes of microbiota were screened at the phylum level. The more detailed analysis was done at the genus-species level, if each microbiota occupied more than 1% of the composition. The statistical analysis was performed using statistical software IBM-SPSS ver 24 [19] unlessotherwise stated. In all statistical analyses, significance was set at P<0.05 unless otherwise stated. A paired t-test was used to detect significant changes, and Spearman's correlation analysis and chief component analysis were done to see interrelation of microbiota for proliferation and inhibition. ASV number and Shannon's index were calculated by R software [20].

Anthropometric Data

All patients who participated in the Ayurveda therapy camp showed improvement in both clinical and mental condition (Table 1). The accompanying doctors checked the state of the patients. Especially diarrhea and dehydration were carefully watched. Meditation, yoga, and health lecture were done in between the therapy.

Bodyweight decreased 1.7 to 5.8 kg (3.7±1.8kg) or 3.2 to 9% (6.4±2.6%) by Virechana therapy (p=0.026), but there was no significant decrease in other methods (Table 2). On the contrary, body fat percentage increased significantly in both Virechana and Basti groups.

Clinical manifestations of participants became better (Figure 3). It was effective in skin rashes by atopic change. The depressed patient had the will to live.

Table 2: Anthropometric change during Ayurveda treatment. The clinical findings decided on the outcome.

*< 0.05

**calculated from "body weight (kg) x 0.4 x 80 kcal)"

Intestinal Microbiota at the Admission of the Camp

At the admission, the most frequent bacteria phylum was Bacteroidetes (46.61±8.90%), Firmicutes (37.5±9.06%), Actinobacteria (2.87±3.02%), Proteobacteria (9.63±4.38%), Cyanobacteria (0.04±0.16%), Fusobacteria (1.68±4.49%), Lentisphaerae (0.01±0.03%), and Synergistetes (0.02±0.09%). Another phylum was not detected above 0.1% (Figure 2).

Everybody possessed Firmicutes, Bacteroidetes, Actinobacteria, and Proteobacteria, but others showed variation; Fusobacteria 33.3%, Verrucomicrobia 46.7%. Cyanobacteria 6.7%, Lentisphaerae, and Synergistetes 13.3% each, Euryarchaeota 6.7%, and Tenericutes 13.3%. At the discharge, Tenericutes increased to 40%, but other phyla returned to the profile at the admission.

Microbiota profile showed different dietary habits pattern, although there was no statistical significance (Table 3 to Table 5). Vegetarians had more Bacteroidetes, Firmicutes, and Actinobacteria than omnivores, who had more Proteobacteria, Fusobacteria, and Cyanobacteria.

Kind of staple foods was divided into polished white rice [5], white rice and barley [4], white rice and brown rice [3], and genmai (brown rice) [3]. Genmai eaters showed higher Firmicutes and less Actinobacteria and Proteiobacteria, but “white rice+barley” group also showed the similar trend. Significant difference among groups was not present.

When the groups were divided by oil consumption, ghee users showed higher Fusobacteriumand less Firmicutes and Actinobacteria (Table 5).

Table 3: Intestinal microbiota by dietary habits at baseline.

Table 3a

Table 3b

Table 3c

Table 4: Intestinal microbiota by rice intake.

Table 5: Intestinal microbiota by habitual intake of ghee.

Changes of Microbiota by panchakarma

Changes by Virechana therapy: The order of top-six microbiota that occupied 99.8% of microbiota at the admission did not change. These were Bacteroidetes (46.6%), Firmicutes (37.5%), Proteobacteria (9.6%), Actinobacteria (2.9%), Fusobacteria (1.7%) and Verrucomicrobia (1.5%). By Virechana, Proteobacteria and Fusobacteria increased to 32.5% and 8.1% respectively. At the discharge time in both Virechana and Basti treated groups, the unassigned bacillus occupied 40.8%, and the Bacteria became 21.1%. These three occupied 93.8%. It was noteworthy that Verrucomucrobia became zero.

Three weeks after the discharge, the composition of the profile returned to the previous pattern. Verrucomicrobia returned to the 6th position.

Treatment-group specific change of microbiota was shown in Table 6. The remarkable changes of microbiota after the pretreatment for Virchana were a decrease of the Firmicutes and an increase of the Proteobacteria (p=0.012 and p=0.023, respectively) (Table 4).

Table 6: Changes of microbiota by Virechana and Basti.

3 Ws later      Admission		Bacteria	Actinobacteria	Bacteroidetes	Cyanobacteria	Firmicutes	Fusobacteria	Lentisphaerae	Proteobacteria	Synergistetes	Bacteriap__TM7	Tenericutes	Verrucomicrobia
Bacteria	P's CC	0.29	0.313	-0.04	-0.1	0.07	-0.2	0.355	-0.01	0.37	.663**	-0	-0.1
	probability	0.29	0.256	0.88	0.67	0.82	0.59	0.194	0.97	0.17	0	0.6	0.8
Actinobacteria	P's CC	0.15	0.444	0.17	-0.2	-0.4	-0.1	-0.12	0.22	-0.1	0.2	-0	-0.1
	probability	0.6	0.097	0.55	0.52	0.1	0.61	0.669	0.44	0.83	0.6	0.4	0.6
Bacteroidetes	P's CC	-0.2	-0.02	0.45	0.25	-.629*	-0.1	-0.43	-0.02	-0.4	0	-0	-.630*
	probability	0.48	0.957	0.09	0.38	0.01	0.77	0.107	0.95	0.13	0.9	0.6	0
Cyanobacteria	P's CC	-0.1	-0.12	0.21	-0.1	0.01	-0.1	-0.09	-0.26	-0.1	-0	-0	-0.1
	probability	0.8	0.664	0.46	0.77	0.97	0.79	0.758	0.36	0.79	0.5	0.7	0.8
Firmicutes	P's CC	-0.1	-0.23	-0.5	0.2	.817**	-0.1	.708**	-0.44	.647**	0.1	0.3	0
	probability	0.75	0.403	0.06	0.48	0	0.7	0.003	0.1	0.01	0.8	0.3	0.9
Fusobacteria	P's CC	.805**	-0.2	-0.14	.649**	-0.1	.734**	-0.12	-0.44	-0.1	-0	-0	-0.1
	probability	0	0.477	0.61	0.01	0.79	0	0.665	0.1	0.7	0.3	0.6	0.8
Lentisphaerae	P's CC	-0.1	-0.03	-0.29	0.01	0.46	-0.1	.700**	-0.34	.983**	0.5	Discussion Ayurveda is a system of medicine with historical roots in the Indian subcontinent [1-4]. Globalized practices derived from Ayurveda traditions are included in a type of alternativemedicine [3,21]. It is said that Ayurveda medicine does not have a scientific basis, so it is often deemed pseudoscientific or trans-science system. Ayurvedic medicine’s central concept is the theory that health exists when there is a balance between the three fundamental bodily bio-elements (doshas) called Vata, Pitta, and Kapha. Panchakarma therapy aims to eliminate excessive Doshas from the body to maintain the state of health for a longer duration. Virechana therapy is one of the Panchakarma therapies wherein detoxication is done by drugs and oil to exclude liposoluble toxins. It specifically aims to eliminate excessive Pitta Dosha from the body. It is said to be beneficial for detoxication in skin disorders, abscesses, and liver disorders because Pitta is considered to control metabolism. Rais S, et al. (2013) [10], conducted the study to evaluate Virechana therapy’s effect on serum electrolytes in 15 people and ascertain the safety of therapeutic purgation. The current study focused on the changes in intestinal microbiota before and after the Panchakarma, and the results were satisfactory to show clinical improvement. Each participant received Virechana, Basti, or milk porridge sudation therapy according to their condition. A personalized plan has carefully scheduled the right selection of oil, sweating, massage, and laxatives or meditation and mindfulness. All symptoms became lighter, especially with skin eruptions. Participants who took part in the trial also got mental settlement to develop a calm mind. All 13 patients showed improvement of skin rash, depression, and other symptoms. The diet in the present camp was the modified Japanese style. They preferred vegetarian food basically, and brown rice eaters showed healthy bowel movement in conjunction with intestinal microbiota [22]. Bodyweight loss seemed to occur only by Virechana therapy, but as the body fat increased in all, the non-fatty mass had also decreased by Basti therapy. Changes in Microbiota and Clinical Manifestation The present participants showed dominancy of Bacteroidetes than Firmicutes. Most Japanese showed dominancy of Firmicutes [22]. It was reflected in their dietary habits, mostly vegetarian way. Vegetarians seemed to have more Bacteroides, Firmicutes, and Actinobacteria than omnivores who had more Proteobacteria, Fusobacteria, and Cyanobacteria. David LA, et al. (2014) [23], analyzed 18 healthy people and found different bacterial profiles by Prakriti (constitution of a person), by Vata, Pitta, and kappa. In this series, Bacteroides, Desulfovibrio, slackia, and succinivibrio were common. Kind of staple foods were five polished white rice, four white rice and barley, three mixtures of white rice and brown rice, and three genmai (brown rice). Genmai eaters showed fewer Actinobacteria and Proteobacteria, but the “white rice+barley” group also showed a similar trend. Significant difference among groups was not present because of the small number of cases. When the groups were divided by oil consumption, ghee users showed higher Fusobacterium and less Firmicutes and Actinobacteria. Panchakarma observed the remarkable changes of the microbiota. After the pretreatment for Virechana therapy, a decrease of Firmicutes and increase of Proteobacteria were noticed. Bacteroides predominated between 47-48% throughout the treatment. The change of microbiota by Basti and Swedana for one week was also the decrease of Firmicutes and increase of Proteobacteria. After the discharge, Firmicutes returned to 30% on average, ranging from 25% to 50%. Proteobacteria returned to baseline level at a range of 4.4% to 8.7% three weeks later. Fusobacterium also increased but no statistical significance. Three weeks after discharge, the variety was increased by Fusobacterium, Verrucomicrobia, Tenericutes, and Lentisphaerae. Hirakawa A, et al. (2019) [12], reported microbiota of 109 healthy people and noted that standard bacterial profiles at the phylum level were 44.3±9.9% Firmicutes, 20.7±8.8% Bacteroides,8.3±6.3% Actinobacteria, 1.7±2.7% Proteobacteria, and 1.2±4.2% (max 39.4%) Verrucobacteria. Their series’s dietary habit was mostly brown rice eaters, so they showed a certain characteristic profile. At the genus level, 12.7% Bacteroides, 8.3% Blautia, 7.9% Faecalibacterium, 6.3% Bifidobacterium, 5.3%Prevotella, 4.9% Eubacterium, 3.8% Ruminococcus, 2.6% Fusicatenibacter, 1.9% Collinsella, 2.4% Streptococcus, 2.1% Subdoligranulum, 1.7% Anaerostipes, 1.2% Akkermansia and 1.7% Roseburia occupied more than 1%. The difference between the brown rice and white rice eaters by microbiota profile was high butyrateproducing bacteria and low fusobacterium. The distribution of butyrate-producing bacteria among Firmicutes phylum seemed to be uneven [25]. Butyrate was not only produced from dietary fiber but lactate. Barley dietary fiber may have a similar effect on microbiota because white rice+Barley showed a similar profile with genmai. The subjective feelings of well having complex determinants and butyrate-producing bacteria could be added among them [12]. We have identified a possible correlation between a high personal sense of health and the presence of butyric acid-producing bacteria in the gut. Besides, increased Enterobacteriaceae in γ-Proteobacteria may produce β-hydroxybutyrate, which yield happy feeling in the brain is a common phenomenon in fasting [26,27]. A weakness of the present study: This was a pre and post therapy study on the relationship between Ayurveda Panchakarma and intestinal microbiota changes. Ayurveda therapy is based upon the tailor-made approach, so it does not fit the ordinary RCT. Accumulation of cases would make new evidence of patient-centered-therapy or narrative medicine. Pre and post-tests would be useful on such occasions [28]. Four significant phyla, Firmicutes, Bacteroidetes, Proteobacteria, and Actinobacteria, were present in all, but other phyla were less current, so the statistical power was insufficient. Decreased Firmicutes and increased Proteobacteria, and loss of Verrucomicrobia were commonly present in all participants, but these changes were usually considered to be worse for health, so more accumulation of clinical cases and long observation is necessary [29]. Changes in bacterial profile would occur by repeated irrigation, which changed the enteroenvironment and many metabolic changes. 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