Immunomodulatory effect of Unani polyherbal formulation - Tiryaq Wabai in mild to moderate COVID-19 patients – A randomized placebo-controlled clinical trial

Abstract

Background

The pathogenesis of COVID-19 includes an integrated immune-inflammatory response. Modulation of host immune responses against the SARS-CoV-2 virus might be effective therapeutic management. Various Unani formulations have an immunomodulatory effect.

Objective

To explore the immunomodulatory effect and safety of Unani polyherbal drug (Tiryaq Wabai) in COVID-19 patients.

Materials and methods

The current study was a randomized placebo-controlled clinical trial that included 92 mild to moderate COVID-19 patients randomized into two groups. The Unani formulation Tiryaq Wabai (2 gm orally once a day) was used as an intervention for 45 days, while the control group received a placebo. Both groups received standard care treatment. The primary outcome was 50% increment in absolute lymphocyte count (ALC). The secondary outcome was 50% increment in mean lymphocyte percentage, CD4 cells, and CD8 cell count. The mean increase in all the above parameters has also been studied. Relevant statistical tests were used to analyze the effect.

Results

A statistically significant improvement in a 50% increase in ALC (p-value, 0.004), lymphocyte percentage (p-value, 0.056), CD4 (p-value, 0.005), and CD8 cell count (p-value, 0.050) was reported. Also, a significant improvement in the mean value of the lymphocyte percentage (p-value 0.0007), ALC (p-value 0.0022), CD4 cell count (p-value 0.0025), and CD8 cell count (p-value 0.0093) was observed after the treatment. One adverse event of mild grade was reported in the placebo group. The analysis of safety parameters (LFT and KFT) was normal for both groups.

Conclusion

In mild to moderate COVID-19 patients, Tiryaq Wabai effectively showed immunomodulatory activity by improving ALC count, lymphocyte percentage, CD4, and CD8 cell count.

Highlights

• •Unani polyherbal formulation (Tiryaq Wabai) possesses potent immunomodulatory properties. The contents of Tiryaq Wabai have antiviral, anti-inflammatory, antipyretic, antioxidant, immunomodulatory, bronchodilator, neuroprotective, anticancer, and anti-mutagenic properties.
• •After 45 days of the treatment, the number of patients with increased ALC, lymphocyte percentage, CD4 cell, and CD8 cell count was significantly higher in the intervention group, compared to the placebo.
• •A significant improvement in the mean value of lymphocyte percentage (p 0.0007), absolute lymphocyte count (p 0.0022), CD4 cells count (p 0.0025), and CD8 cells count (p 0.0093) was observed after the treatment.
• •No significant adverse events or deranged LFT, KFT test values were reported for the intervention group.

1. Introduction

The COVID-19 pandemic caused by the SARS-CoV-2 virus has had serious consequences for the global economy and healthcare systems. Since its emergence in December 2019 in Wuhan, city of China, it has affected almost every country across the world, with 626 million global cases, including 6.56 million deaths [1]. The incubation period reported for the SARS-CoV-2 virus was 2.1–11.1 days, with an average of 6.4 days [2]. Patients have a diverse range of clinical indications and severity, from being asymptomatic, normal flu-like illness to having a severe interstitial pneumonia-like condition that may lead to ARDS, fatal hypoxemia, multi-organ failure, and death [3]. A more severe form of COVID-19 has been reported in the elderly, immune-compromised individuals, and patients with underlying medical comorbidities [4].

SARS-CoV-2 is positive-sense single-strand RNA virus. It has the largest genome of all RNA viruses and contains spike (S), membrane (M), nucleocapsid (N), and envelope (E) proteins [5]. For virus replication, about 16 nonstructural proteins (NSP): proteases, RNA-dependent RNA polymerases, nuclease, helicase, and transferase are encoded through 14 open reading frames (ORFs). This virus affects the innate immunity of the host, especially the type 1 Interferon response. An integrated immune-inflammatory reaction known as a cytokine storm is the chief pathogenesis caused by SARS-CoV-2. It leads to the release of inflammatory cytokines, e.g., IFN-γ, tumor necrosis factor-α, interleukin-1β, interleukin-2, interleukin-6, and interleukin-7 [6].

The suggested immune response against COVID-19 involves the stimulation of B-lymphocyte cell and T-lymphocyte cell epitopes that stimulate CD4 and CD8 T-cells to aggravate antibody-mediated and cell-mediated immune responses [7]. Decreased T-lymphocytes, CD4, and CD8 cell counts were reported in COVID-19. In most severe cases, the number of B-cells, CD4 cells, CD8 cells, and NK cells decreases. Patients responding to treatments have increased CD8 T and B cells, unlike those who didn't respond. Evidence from SARS-CoV animal studies suggests that decreased CD4 T-cells may lead to decreased lymphocyte recruitment, cytokine production, and neutralizing antibody titers [4].

Treatments for COVID-19 are divided into three groups: medications that block virus entry into the host cell, medications that block viral replication and endurance inside the host cell, and medications that modulate the host immune response [2]. Together with the specific therapy directed straight against COVID-19, the enhancement of host immune responses against the virus might play a vital role in its management. Primary management includes antipyretic, anti-inflammatory, antiviral, anti-asthmatic, and immunomodulatory drugs. Immunomodulation refers to an alteration in the immune response that includes inhibition, initiation, expression, and intensification of any phase of the immune response [8].

The basis of the Unani system of medicine is the concept of temperament (mizaj) and four humours (akhlat) described by Hippocrates. The human body consists of mainly four humours, e.g., phlegm, sanguine, black bile, and yellow bile [9]. The symptoms of COVID-19 are equivalent to ‘Nazla wa zukaam wabayi’ (epidemic influenza) and Humma wabai (epidemic fever) as described by Ajmal Khan. The concept and management of epidemic illness (Amraz Wabaiyah) have been described in various Unani treatises like Al-Qanun fil Tib (Canon of Medicine), Kamil-Us-Sana (Liber regius), Zakhira Khqwazam Shaahi (Treasure of Khvarazm Shah), Akseer-e-Azam (Al-Akseer), Al-Havi (Liber continence), Hummiyat-e-Qanoon, Kitabul Mansoori, and Kitab Al-Taisir [10]. In Unani literature, various herbs have been described as possessing antiviral and immunomodulatory properties.

The single herbal drugs reported with immunomodulatory activity are Elwa (Aloe vera), Gilo (Tinospora cordifolia L.), Asgand (Withania somnifera L.), Kalonji (Nigella sativa), turmeric (Curcuma longa), Zanjabeel (Zingiber officinale), Aslussus (Glycyrrhiza glabra L.), Filfil daraz (Piper longum L.), Halela (Terminalia chebula Retz.), Amla (Emblica Officinalis L.), Rehan (Ocimum sanctum), cinnamon (Cinnamomum zeylanicum), Kath (Acacia catechu), Musli safed (Chlorophytum borivilianum), Amaltas (Cassia fistula L.), Saffron (Crocus sativus L.), Shatavari (Asparagus racemosus Wild), Behi dana (Cydonia oblonga), Unnab (Zizyphus jujuba), and Karanjwa (Caesalpinia bonducella) [5,[11], [12], [13]]. The compound formulations with antiviral and immunomodulatory properties are Triyaq Araba (detoxifying agent, anti-viral), Tiryaq Wabai (anti-inflammatory, antioxidant, analgesic, immunomodulatory), Laooq Sapistan, Sharbat Sadar, and Khamira Banafsha (for cough and respiratory disorders), Khamira Marwarid (immunomodulator, anti-viral), and Sharbat Toot Siyah (sore throat, anti-inflammatory, analgesic, immunomodulatory) [5,12,14,15].

Based on the above-mentioned evidence, this clinical trial was done to explore the immunomodulatory effect of the Unani polyherbal drug (Tiryaq Wabai) in COVID-19 patients.

2. Methodology

2.1. Trial design

This was a parallel-group, randomized, placebo-controlled clinical study. The chronologically numbered, sealed envelope method was used for patient allocation to treatment groups. A computer-generated allocation series were used for randomization. All the patients, the staff organizing the trial medicine, the treating clinicians, and the investigators reporting outcome data and conducting the result analyses were blind to allocation. A single unblinded staff member was recruited to provide trial medication and maintain protocol adherence and violations.

2.2. Site of the study

The study was conducted from January 2021 to February 2022 at Rajendra Institute of Medical Sciences (RIMS), located in Ranchi, Jharkhand, India, after obtaining ethical approval from the Institutional Ethics Committee, RIMS, Ranchi (ECR/769/INST/2015/RR-18) reference No. 018 dated May 15, 2020.

2.3. Participants

A total of 92 patients with mild to moderate COVID-19 disease willing to participate were recruited after receiving written informed consent about their participation. The complete procedure of the study was explained to all the participants in their own language by the investigator before recruitment.

2.4. Inclusion criteria

• 1.Patients aged between 18–75 years.
• 2.RT-PCR positive COVID-19 patients.
• 3.American Society of Anesthesiologists (ASA) classes I and II [16]. ASA Class I includes a normal, healthy patient, e.g., a fit, nonobese (BMI under 30) nonsmoking patient with good exercise tolerance. ASA Class II includes patients with mild systemic disease, e.g., patients with no functional limitations and a well-controlled disease (e.g., treated hypertension, obesity with a BMI under 35, frequent social drinkers, or cigarette smokers).
• 4.Patients with co-morbid conditions, e.g. controlled hypertension and diabetes mellitus.

2.5. Exclusion criteria

• 1.Suspected COVID-19 patient.
• 2.Pregnant and lactating women.
• 3.History of hypersensitivity reactions to the study drug or any of its constituents.
• 4.Patients with neuropsychiatric disorders.

2.6. Sample size

The sample size for this trial was calculated based on the results of a pilot study carried out at RIMS, Ranchi. That pilot study was not published. The proportion of patients with a 50% increase in ALC was found to be 70% and 40% in the intervention and control groups, respectively; the calculated sample size became 40 per group. After the addition of a 15% loss to follow-up, the recommended sample size was calculated as 46. Thus, a total of 92 patients were enrolled in this study. The calculation was performed using the select statistical consultants website's online calculator tool named ‘Comparing two proportions: sample size’ [17].

2.7. Trial interventions

This trial was conducted on the Unani polyherbal formulation Tiryaq Wabai. The intervention drug and its placebo were procured from GMP (Good Manufacturing Practice) certified Unani medicine manufacturer Liimra (U&A) Remedies, Haryana (India). The intervention group was treated with Tiryaq Wabai, a polyherbal formulation with a semisolid paste-like consistency. This drug was given 2g orally, once a day, in the morning before breakfast (with normal water only if necessary) for a period of 45 days. The same dose of a wheat-based placebo with similar consistency and appearance was administered to the control group for 45 days. The first 10 days of this study were part of a COVID-19 trial with the Unani formulations Tiryaq Wabai, Arq-e-Ajeeb, and Habb-e-Loban used as an adjuvant drug to standard care treatment in the intervention group. In the control group, placebo for Tiryaq Wabai, Arq-e-Ajeeb, and Habb-e-Loban was given at a similar dose and duration. Both groups received standard care treatment for COVID-19 for the first 10 days only based on the recommendations of the Ministry of Health and Family Welfare (MoHFW), Government of India. These included antipyretics, proton pump inhibitors, multivitamins with zinc, vitamin C, antibiotics (Azithromycin, Doxycycline, and Ivermectin), and anti-allergic medications, along with oxygen support.

Tiryaq Wabai, also known as Tiryaq-e-Afai and Habb-e-Taaun [15], has been widely used during epidemics for management and prophylaxis against cholera, plague, smallpox, and other epidemic diseases since the ancient era by Unani scholars Ibn Sina (Avicenna, 980–1035 AD) and Jalinoos (Galen, 131–199 AD) [18]. As per the Unani pharmacopoeia (Bayaz Kabir) [19], Tiryaq Wabai is composed of three ingredients, i.e., dried juice of leaves of Aloe barbadensis Mill., gum resin of Commiphora myrrha., and dried style and stigma of Crocus sativus Linn., in the proportion of 2:1:1.

2.8. Assessment and follow-up of the participants

After inclusion in the study, demographic data, baseline clinical findings, date of admission to the hospital, chief COVID-19 symptoms with their duration before hospital admission or inclusion, and associated co-morbidities were recorded. Any relevant ECG, chest X-ray, or HRCT-lung findings were noted. The assessment of absolute lymphocyte count (ALC), total leucocyte count (TLC), lymphocyte percentage, CD4 cell count, and CD8 cell count was done on days 1, 15, and 45. Any special investigations were performed as per the comorbidity present in the case. After discharge from the hospital, follow-ups were done every 15 days, i.e., on the 15th, 30th, and 45th days. Any adverse effects such as nausea, vomiting, lacrimation, itching, etc. were noted and reported. All the data were documented in case report form (CRF) designed for the project and in Excel sheets for analysis purposes. Any protocol violations, e.g., use of other herbal medications and irregular administration of trial medication, were recorded.

2.9. Outcomes

The proportion of patients with a 50% increase in mean absolute lymphocyte count (ALC) was the primary outcome. Mean changes in the laboratory parameters, e.g., WBC count, lymphocyte percentage, absolute lymphocyte count, CD4 and CD8 cell counts, and CD4/CD8 ratio, were also analyzed. The secondary outcomes were (i) the proportion of patients with a 50% increase in lymphocyte percentage on days 15 and 45; (ii) the proportion of patients with a 50% increase in CD4 cell count on days 15 and 45; (iii) the proportion of patients with a 50% increase in CD8 cell count on days 15 and 45; (iv) the percentage of patients with adverse events.

2.10. Statistical analyses

Intention-to-treat (ITT) analysis was done for both primary and secondary outcomes. The statistical software IBM SPSS version 24 was used for data analysis. The data with categorical variables were expressed as numbers and percentages, while the continuous variables were expressed as the mean ± SD. Paired t-tests and unpaired t-tests were used for analyzing continuous variables in intra-group and inter-group analyses, respectively. The Fisher's exact test and Pearson's chi-square were used for analyzing categorical variables. The p-values of <0.05 were considered to be significant.

3. Results

A total of 272 suitable patients were screened, out of whom 79 were denied participation. The reason for screening a larger number of patients was that the majority of patients admitted to the hospital were opposed to combining herbal treatment with standard care treatment due to a lack of information and faith in herbal medicine. A few patients denied taking trial medicines, even after providing formal consent. One hundred and one patients were excluded due to associated co-morbidities or certain medical conditions. Ninety-two patients were randomized into two groups and allocated into the intervention and control groups. One patient discontinued treatment after four days of inclusion. Five patients failed to complete follow-up (see Fig. 1). Intention-to-treat analysis was done by using the last-observation-carried-forward (LOCF) method for missing data handling.

Fig. 1.

CONSORT flow diagram

3.1. Clinico-demographic result

Table 1 represents demographic characteristics like age, gender, BMI, and baseline laboratory values for both groups. Fever, cough, weakness, breathlessness, and chest pain were the most prominent symptoms in COVID-19 patients.

Table 1.

Baseline clinico-demographic characteristics

Variables	Control Group (n = 46)	Intervention Group (n = 46)	P-value
Demographic Characteristics:
Male sex (n, %)	31	33	0.650a
Age in years (mean ± SD)	50.13 ± 15.02	48.78 ± 15.68	0.674b
BMI (mean ± SD)	25.69 ± 2.47	25.47 ± 2.60	0.668b
Duration of symptoms in days before enrollment (mean ± SD)	5.91 ± 3.37	6.49 ± 3.07	0.398b
Comorbidities (n, %)	11 (23.9)	9 (19.6)	0.801a
Prominent COVID-19 symptoms:
Fever	38	36	0.793 a
Cough	36	38	0.793 a
Weakness	17	22	0.398 a
Breathlessness	17	20	0.671 a
Chest pain	10	13	0.631 a
Body ache	9	7	0.784 a
Altered smell sensation	5	7	0.758 a
Altered taste sensation	3	4 (	>0.999 a
Laboratory Parameters:
WBC, (103/μL) mean ± SD	7.119 ± 3.87	7.001 ± 2.98	0.8703b
Lymphocyte Percentage, mean ± SD	20.67 ± 9.82	18.09 ± 9.53	0.203b
Absolute Lymphocyte Count (103/μL), mean ± SD	1.32 ± 0.77	1.12 ± 0.57	0.157 b
CD4 Cell Count (cell/μL), mean ± SD	422.04 ± 328.69	388.30 ± 242.28	0.576 b
CD8 Cell Count (cell/μL), mean ± SD	348.17 ± 223.80	359.39 ± 218.97	0.809 b

^aFisher's exact Test.

^bUnpaired T-Test.

3.2. Outcome analysis

An analysis of primary and secondary outcomes is mentioned in Table 2 and is represented graphically in Fig. 2. After 45 days of treatment, the number of patients with increased ALC, lymphocyte percentage, CD4, and CD8 cell counts was significantly higher in the intervention group. The Chi-Square or Fisher's exact test was used for analysis.

Table 2.

Analysis of primary and secondary outcomes

Sr. No	Outcome Variables	Intervention (n = 46)	Control (n = 46)	P valuea
1.	Number of patients with a 50% increase in mean ALC on Day 15	33	16	0.0004b
2.	Number of patients with a 50% increase in mean ALC on Day 45	37	24	0.0041b
3.	Number of patients with a 50% increase in mean lymphocyte percentage on Day 15	17	09	0.104
4.	Number of patients with a 50% increase in mean lymphocyte percentage on Day 45	24	14	0.056
5.	Number of patients with a 50% increase in mean CD4 at Day 15	29	20	0.06
6.	Number of patients with a 50% increase in mean CD4 at Day 45	39	27	0.005b
7.	Number of patients with a 50% increase in mean CD8 at Day 15	22	17	0.2915
8.	Number of patients with a 50% increase in mean CD8 at Day 45	34	25	0.050

^aChi-Square/Fisher's exact test is used.

^bStatistically significant.

Fig. 2.

Analysis of primary and secondary outcomes

Table 3 shows the effect of the intervention drugs on laboratory parameters. There was a significant improvement in lymphocyte percentage (p 0.0007), absolute lymphocyte count (p 0.0022), CD4 cell count (p 0.0025), and CD8 cell count (p 0.0093) at day 45 of the treatment. No significant differences were found in WBC count (p 0.9495), CD4 cell percentage (p 0.3947), CD8 cell percentage (p 0.2046), or CD4/CD8 ratio (p 0.8609) after treatment.

Table 3.

Analysis of laboratory parameters.

Parameters	Time Point	Intervention Group (n = 46) (Mean ± SD)	Control Group (n = 46) (Mean ± SD)	Between Group (p-value)a
WBC (103/μL)	Baseline	7.001 ± 2.98	7.119 ± 3.87	0.8703
	Day 15	8.155 ± 2.01	8.052 ± 2.69	0.8359
	Day 45	7.902 ± 1.67	7.936 ± 3.235	0.9495
Lymphocyte Percentage	Baseline	18.09 ± 9.54	20.68 ± 9.83	0.2032
	Day 15	23.74 ± 8.04	22.13 ± 8.78	0.3594
	Day 45	28.87 ± 7.517	23.59 ± 6.87	0.0007b
Absolute lymphocyte count (103/μL)	Baseline	1.121 ± 0.57	1.327 ± 0.77	0.1507
	Day 15	1.893 ± 0.67	1.676 ± 0.73	0.1404
	Day 45	2.338 ± 1.02	1.788 ± 0.60	0.0022b
CD4 Cell count (cell/μL)	Baseline	388.3 ± 242.3	422.0 ± 328.7	0.5766
	Day 15	632.4 ± 313.7	581.4 ± 327.3	0.4481
	Day 45	825.2 ± 344.6	628.0 ± 258.1	0.0025b
CD8 Cell count (cell/μL)	Baseline	359.4 ± 219.0	348.2 ± 223.8	0.8086
	Day 15	523.0 ± 265.4	447.8 ± 223.6	0.1454
	Day 45	665.9 ± 358.8	502.7 ± 211.3	0.0093b
CD4 Percentage	Baseline	33.68 ± 9.45	33.02 ± 12.65	0.7766
	Day 15	35.48 ± 9.49	36.21 ± 11.35	0.7409
	Day 45	38.70 ± 8.83	37.09 ± 9.19	0.3947
CD8 Percentage	Baseline	32.11 ± 11.37	28.52 ± 9.64	0.1060
	Day 15	29.63 ± 8.32	28.65 ± 8.42	0.5790
	Day 45	31.47 ± 8.29	29.40 ± 7.19	0.2046
CD4/CD8 Ratio	Baseline	1.216 ± 0.73	1.232 ± 0.49	0.9034
	Day 15	1.248 ± 0.36	1.327 ± 0.47	0.3749
	Day 45	1.306 ± 0.37	1.321 ± 0.45	0.8609

^aBetween-group p-value, compared using an independent sample t-test/Mann-Whitney test.

^bStatistically significant.

3.3. Analysis of safety parameters and adverse events

No significant differences in kidney function tests (urea and creatinine) and liver function tests (SGOT, SGPT, total bilirubin, alkaline phosphatase level) were reported between the two groups (see Table 4). One patient in the placebo group discontinued the trial drug due to itching that began after four days of treatment. No complications, e.g., nausea, vomiting, ARDS, sepsis, arrhythmia, etc., were reported in either group.

Table 4.

Analysis of safety parameters.

Parameters	Time Point	Intervention Group (n = 46) (Mean ± SD)	Control Group (n = 46) (Mean ± SD)	Between Group (p-value)a
Blood Urea (mg/dL)	Baseline	32.56 ± 15.66	30.26 ± 15.85	0.485
	Day 10	32.14 ± 12.51	28.95 ± 12.58	0.226
	Day 45	29.26 ± 11.49	26.90 ± 10.25	0.304
Serum Creatinine (mg/dL)	Baseline	1.01 ± 0.26	0.98 ± 0.26	0.581
	Day 10	0.95 ± 0.26	0.97 ± 0.23	0.716
	Day 45	0.83 ± 0.21	0.93 ± 0.27	0.047
SGOT/AST (IU/L)	Baseline	52.89 ± 40.18	45.25 ± 26.46	0.284
	Day 10	47.99 ± 28.62	39.61 ± 15.51	0.084
	Day 45	40.28 ± 15.56	35.17 ± 13.60	0.096
SGPT/ALT (IU/L)	Baseline	47.86 ± 35.22	46.27 ± 34.72	0.827
	Day 10	46.59 ± 29.17	42.12 ± 26.15	0.441
	Day 45	39.87 ± 27.94	32.65 ± 12.17	0.111
Total Bilirubin (mg/dL)	Baseline	0.64 ± 0.28	0.75 ± 0.41	0.136
	Day 10	0.69 ± 0.31	0.68 ± 0.27	0.834
	Day 45	0.60 ± 0.28	0.62 ± 0.23	0.767
Alkaline Phosphatase (mg/dL)	Baseline	93.42 ± 43.83	90.19 ± 35.73	0.699
	Day 10	90.98 ± 42.70	92.24 ± 31.45	0.872
	Day 45	80.74 ± 26.16	85.96 ± 23.69	0.318

^aBetween-group p-value, compared using independent sample t-test/Mann-Whitney test. SGOT: serum glutamic oxaloacetic transaminase; SGPT: serum glutamic pyruvic transaminase.

4. Discussion

This clinical trial aimed to explore the immunomodulatory effect of a polyherbal Unani formulation (Tiryaq Wabai) in mild to moderate COVID-19 patients. Another two drugs, given only for a preliminary period of 10 days, may have had a very minimal additive effect on immunomodulation. Arq-e-Ajeeb has mainly anti-allergic and anti-asthmatic effects. Habbe-e-Loban possesses anti-inflammatory and expectorant properties and is used for cough, pneumonia, and pleurisy. A significant improvement was observed in the intervention group for most of the studied outcomes, like the proportion of patients with a 50% increase in mean ALC, lymphocyte percentage, CD4, and CD8 cell count. Analysis of mean ± SD values of laboratory parameters showed a significant improvement in lymphocyte percentage, absolute lymphocyte count, CD4 cell count, and CD8 cell count at day 45 of the treatment. The active phytoconstituents and metabolites of this Unani formulation are to be accredited for that effect.

The three herbal ingredients of the test drug Tiryaq Wabai are Crocus sativus, Aloe barbadensis, and Commiphora myrrh. Saffron (Crocus sativus) possesses antiviral, antioxidant, bronchodilator, anti-inflammatory, neuroprotective, anticancer, and anti-mutagenic properties [10,[20], [21], [22]]. It is used in the management of bronchitis, asthma, colds, catarrh, coryza, fevers, diabetes, neurological disease, and coronary artery disease. The major biologically active substances in saffron are safranal, kaempferol, monoterpene aldehydes, alpha and beta carotene, crocin, comprol, pyrocrocin, and crocetin [23,24].

Various studies reported that bioactive constituents of saffron possess an immune-boosting effect and modulate both acquired immunity (anti-inflammatory and inflammatory cytokines levels, B-lymphocyte cells, and Type 1/Type 2 helper cells) and innate immunity (neutrophils, macrophages, and NK cells) [23]. Animal studies reported that safranal reduces the levels of TNF-α, IL-1β, and IL-4 levels, stimulates IFN-γ, IL-10 levels, and IFN-γ/IL-4 ratio, and modulates the Th1/Th2 proportion [23,24]. A study on mice infected with the toxin of Pasteurella multocida reported that oral intake of C. sativus (50–100 mg per kg) enhanced the overall humoral-mediated immunity through the amplification of the immunoglobulin, circulating antibody, and adhesion molecules of neutrophils [25]. Other studies on mice reported that the extracts of saffron decreased the amount of reactive oxygen species and pro-inflammatory nitric oxide production. The extract also improved catalase (CAT) and superoxide dismutase levels [26,27]. A clinical trial with 45 healthy cases reported that an intake of 100 mg of saffron for 42 days decreased IgM and increased IgG and monocytes when correlated to the baseline and placebo groups [28].

Crocin modulates the humoral and cellular immune systems and decreases TNF-α, NF-κB, IL-6, IL-8, and IL-1β levels [24]. An animal study reported that saffron constituent crocin used orally at a dose of 9.69 mg/kg improved cellular immunity by increasing the adhesion of neutrophils and phagocytic index, and reducing cyclophosphamide-induced neutropenia [29]. Crocetin also showed immunomodulatory effects through the reduction of NF-κB, IL-6, and IL-8 levels, and inhibition of inflammatory cytokine production (TGF-β1 and TNF-α) [24]. An animal study on mice reported that the flavonoid kaempferol found in saffron significantly reduced IFN-γ production [30] and the manifestation of TNF-α and IL-4 at mRNA [31]. Also, it prevents nuclear factor-inducing kinase (NIK)/IκB kinase (IKK) activation and MAPKs signaling pathways, thus hindering NF-κB function [32]. This revealed that saffron has a great effect on immunomodulation and the management of COVID-19.

Dried pulp from the leaves of Aloe barbadensis (Aloe) is the second constituent of the intervention drug Tiryaq Wabai. About 200 potentially active constituents are present in aloe vera, which are responsible for its multifunctional properties. It possesses immunomodulatory, antiviral, anti-inflammatory, antipyretic, antioxidant, anti-asthmatic, anti-aging, antiseptic, and laxative activities [[33], [34], [35], [36], [37]]. Aloe-emodin and anthraquinone aloin found in aloe have been reported to be effective in the deactivation of several viruses like HSV types I and II, influenza virus, and varicella zoster [38]. Aloe vera modulates the intestinal macrophages, permitting the immune system to improve T-lymphocyte activity by more than 50% to kill various pathogens, including bad bacteria, viruses, and tumor cells [39]. Acemannan (a polysaccharide) found in aloe is an antiviral agent. A clinical study reported its immune enhancement capabilities and dose-dependent increase in killer T-cell production [40]. Lectin present in aloe vera functions as a mitogenic agent and enhances the frequency of lymphocyte proliferation and growth. It also facilitates the recognition of foreign bodies and cells and the stimulation of endocytosis through macrophages. An animal study on mice by Gonzalez et al. reported enhanced proliferation of T-lymphocytes in the involuted thymus when treated with a 30 mg water extract of Aloe Vera [41].

The third constituent of the intervention drug is the gum resin of Commiphora myrrh (Mur). It contains various flavonoids, alkaloids, tannins, glycosides, steroids, saponins, tannins, and terpenoids. Studies have reported the antioxidant, antimicrobial, anti-inflammatory, analgesic, neuroprotective, antiviral, and hepatoprotective effects of Commiphora myrrh and its effectiveness for chronic cough, asthma, bronchitis, the common cold, etc. [15,42]. Commiphora myrrh activates later stages of the reproductive and maturation stages for different types of leukocytes in the active phase of specific immune reactions. It helps regulate a relative increase in the number of leukocytes during the recovery period [43].

The increase in absolute lymphocyte count (ALC), lymphocyte percentage, CD4 cell count, and CD8 cell count after using Tiryaq Wabai is supported by another study by Nigar Z, where a similar improvement was reported in elderly patients [44].

Therefore, this Unani formulation includes active phytochemicals for COVID-19 management with the help of anti-viral activity and immune modulation. Network pharmacological methods can be a valuable tool to understand the effects, indications, and science-based mechanisms of these Unani medicines for COVID-19. Besides one adverse event of the mild category in the placebo group, the study did not report any other adverse events, e.g., GIT symptoms (nausea, vomiting, anorexia), or derangements in liver function tests or renal function tests. Also, the Unani formulation was safe to be used with modern medicine with no drug-drug interactions.

4.1. Limitations of the study

Only COVID-19 patients with mild-to-moderate severity were recruited for this study; therefore, the findings of this study cannot be applied to severe COVID-19 patients. Several cytokines also have crucial roles in the immune response, which consist of pro-inflammatory cytokines (e.g., TNF-α, IL-1, IL-6, IL-11, IL-8), anti-inflammatory cytokines (IL-4, IL-10, and IL-13), C-reactive protein, procalcitonin, and D-dimer, which were not assessed.

5. Conclusions

The Unani polyherbal formulation Tiryaq Wabai significantly increased the absolute lymphocyte count (ALC), lymphocyte percentage, CD4, and CD8 cell counts in COVID-19 patients with mild to moderate severity after 45 days of treatment. No significant adverse effect was reported in either group. Also, these drugs are safe to use with modern allopathic treatment. A clinical trial with a large sample size may be conducted to validate the study. The intervention drug might be studied in other viral diseases or immune-compromised disorders.

Funding

This work was funded by the Ministry of AYUSH, Government of India.

CRediT author statement

AK: Conceptualization, methodology, investigation, supervision, funding acquisition, writing – review & editing. MSK: Investigation, data curation, formal analysis, writing – review & editing. ZUH: Investigation, data curation, formal analysis, writing - original draft, review & editing. AR: Methodology, formal analysis, visualization, writing – review & editing. MF: methodology, visualization, GR: Visualization. All authors read and approved the final draft. All data was generated in-house, and no paper mill was used. All authors agree to be accountable for all aspects of their work, ensuring integrity and accuracy.

Declarations of any conflicts of interest

No potential conflicts of interest were reported.

Trial registration

The registration of the study in the Clinical Trial Registry of India (CTRI) was done before the recruitment of the patient (Registration No. CTRI/2021/01/030825).

Declaration of competing interest

No potential conflicts of interest were found concerning the research study, authorship, or article publication.