Pharmacokinetics of a Natural Self-emulsifying Reversible Hybrid-Hydrogel (N’SERH) Formulation of Full-Spectrum Boswellia serrata Oleo-Gum Resin Extract: Randomised Double-Blinded Placebo-Controlled Crossover Study

Ashil Joseph; Maliakkal Balakrishnan Abhilash; Johannah Natinga Mulakal; Krishnakumar Illathu Madhavamenon

doi:10.1248/bpb.b24-00306

Abstract

The oleo-gum-resin of Boswellia serrata, an Ayurvedic herb for the treatment of chronic inflammatory diseases, contains both volatile (terpenes) and nonvolatile (boswellic acids) molecules as responsible for its bioactivity. The present randomized, double-blinded, placebo-controlled, crossover study evaluated the human pharmacokinetics of a ‘natural’ hybrid-hydrogel formulation of a unique full-spectrum boswellia extract (BFQ-20) (standardized for both volatile and nonvolatile bioactives) in comparison with unformulated extract (U-BE), for the first time. Mass spectrometry coupled with LC (UPLC-MS/MS) and gas chromatography (GC-MS/MS) measurements of the plasma concentration of boswellic acids and α-thujene at different post-administration time points followed by a single dose (400 mg) of U-BE and BFQ-20, to healthy volunteers (n = 16), offered 4-fold enhancement in the overall bioavailability of boswellic acids from BFQ-20, [area under the curve (AUC) (BFQ-20) = 9484.17 ± 767.82 ng * h/mL vs. AUC (U-BE) = 2365.87 ± 346.89 ng * h/mL], with the absorption maximum (T_max) at 6.3 h post-administration and elimination half-life (T_1/2) of 15.5 h (p < 0.001). While plasma α-thujene was not detectable upon U-BE administration, BFQ-20 provided significant absorption, [AUC (BFQ-20): 298.60 ± 35.48 ng * h/mL; C_max: 68.80 ± 18.60 ng/mL; T_max: 4.12 ± 0.38 h; T_1/2: 16.24 ± 1.12 h]. Further investigation of the anti-inflammatory effect revealed 70.5% inhibition of paw edema in rats compared to 38.0% for U-BE. In summary, the natural self-emulsifying reversible hybrid-hydrogel (N’SERH) formulation of boswellia extract using fenugreek mucilage (FenuMat^®) significantly increased the solubility (58-fold), stability, and bioavailability of both the volatile and non-volatile bioactives which in turn improved the anti-inflammatory efficacy of Boswellia extract.

INTRODUCTION

Boswellia serrata (Indian Frankincense or Salai guggul) is one of the most valued Indian medicinal plants belonging to the family Burseraceae. The oleo-gum resin obtained from the barks has been a promising traditional remedy for chronic inflammatory diseases such as arthritis, bronchitis, asthma, ulcerative colitis, atherosclerosis, liver cirrhosis, and even cancer.^1–3) Boswellia serrata (B. serrata) gum resin contains more than 200 different compounds like terpenes, terpenoids, carboxylic acids, and polyphenols,^4–6) in which boswellic acids (BAs), chemically characterized as a group of pentacyclic triterpenoid carboxylic acids resembling steroidal anti-inflammatory compounds, have been identified as the key bioactive molecules.^7,8) Based on the chemical structure, six major boswellic acids have been characterised, including α- and β-boswellic acids (αBA and βBA, 10–21%), acetylated α- and β-boswellic acids (AαBA and AβBA, 0.05–6.0%), 11-keto-β-boswellic acid (KBA, 2.5–7.5%) and 3-O-acetyl-11-keto-β-boswellic acid (AKBA, 0.1–3%).^1,2) The oleo-gum-resin is also rich in oil (volatile and non-volatile oils, 8–15%), in which α-thujene (>60%) has been identified as the main bioactive molecule.⁹⁾

Supplementation of boswellia extracts standardised for BAs has been shown to have potential effects on several molecular targets involved in the pathogenesis of inflammation, such as 5-lipoxygenase (5-LOX), cyclooxygenase-1 (COX-1), nitric oxide inhibition,¹⁰⁾ mitogen-activated protein kinase (MAPK), microsomal prostaglandin-E synthase (mPGES)-1, Cathepsin-G, nuclear transcription factor κB (NF-κB), pro-inflammatory cytokines like tumour necrosis factor α (TNFα), interleukin (IL)-1β, IL-2, IL-6 and histamines.^11,12) Previous reports based on both in-vitro and in-vivo studies could establish the anti-cancer potential of BAs at various stages of cancer progression; including the prevention of cell proliferation, metastasis, invasion, migration, or other cellular processes related to cancer.^3,7,8) Antiplatelet aggregation, anti-profibrotic effect, gastroprotective effect, anti-neurodegenerative effect, hepatoprotective effect and amelioration of oxidative stress have also been reported for the extracts of boswellia gum resin.^13–15) A recent molecular docking and an in-vitro study demonstrated that β-boswellic acids have strong inhibitory effect against Severe acute respiratory syndrome coronavirus 2E (SARS-CoV-2E) protein inhibition.¹⁵⁾ Boswellia extracts have also been shown to inhibit the expression of pro-inflammatory cytokines from immune-competent cells and eventually prevents insulitis and insulin resistance in type 1 and type 2 diabetics.¹⁶⁾ Interestingly, the oil extracted from boswellia oleo-gum-resin devoid of boswellic acids have also shown anti-inflammatory, antimicrobial, anticancer effects.^17,18) So, the medicinal properties of boswellia oleo-gum resin were considered to originate from the synergistic effect of the non-volatile boswellic acids and volatile terpenes and terpenoids from its essential oil.

Pharmacokinetic investigations of boswellia extracts standardised to various levels of boswellic acids, in both animals and human volunteers, have revealed very low plasma and tissue concentrations of BAs even at high doses (3 g/d).^19–21) Among the various boswellic acids, systemic concentration of the most active KBA and AKBA were found to be extremely low.²⁾ The very low absorption and bioavailability of BAs may be attributed to its hydrophobicity, poor aqueous solubility, extensive metabolism, and low permeability.^12,22) To overcome these bioavailability barriers, various microencapsulation approaches and nanoemulsions with synthetic surfactants have been developed.^23,24) Though many of these techniques may find applications in pharmaceutical drug delivery, their usage in food and nutraceuticals have been seriously limited by regulatory barriers, especially due to the extensive usage of synthetic emulsifiers, excipients, and nano form. Moreover, no study has been conducted on the pharmacokinetics of the volatile fractions of boswellia resin gum extracts. This is due to the difficulty in developing stable formulations of boswellia extracts standardised to contain both volatile (essential oil) and non-volatile bioactive molecules.

In the present study, we characterised and further investigated the human pharmacokinetics of a full-spectrum formulation of B. serrata oleo-gum resin extract standardised to contain both volatile (essential oil) and non-volatile bioactive fractions. The formulation was developed as a self-emulsifying hybrid-hydrogel using the naturally occurring fenugreek seeds mucilage (galactomannan) and sunflower lecithin. The formulation (hereinafter denoted as ‘BFQ-20’) was a water dispersible powder which can swell into a hydrogel and emulsify under gastrointestinal conditions. Recently we have reported such hybrid-hydrogel delivery forms of phytonutrients like trans-resveratrol, quercetin, fisetin and vitamin C with improved solubility and bioavailability upon oral administration.^25–28)

MATERIALS AND METHODS

Preparation of Extracts

Standardised extract of B. serrata oleo-gum resin (U-BE) and its hybrid-hydrogel formulation (BFQ-20) were manufactured and provided by Akay Natural Ingredients, Kochi, India, following good manufacturing practices (GMP). U-BE was prepared by a hydro-ethanolic extraction, followed by evaporation, concentration and drying. BFQ-20 was prepared by a proprietary gel-phase thin-film dispersion process to impregnate boswellia extract emulsions into the galactomannan hydrogel matrix, followed by evaporation to powder form. A detailed certificate of analysis, material safety data sheet and declaration of its food-grade and clean label status (non-genetically modified, allergen-free, vegan, and absence of synthetic emulsifiers, excipients, and polymers) was also received from the manufacturer.

The BAs content in the extracts was analysed by a validated HPLC method,²⁹⁾ employing the Shimadzu LC 20AT instrument fitted with an M20A photodiode array detector (PDA) (Shimadzu Corporation, Kyoto, Japan) and reverse phase C18 column (250 × 4.6 mm, 3 µm). The α-thujene content was determined by gas chromatography (GC) coupled with triple quadruple mass spectrometer (MS/MS) (Shimadzu Nexis GC-2030), employing a polysiloxane column (Shimadzu Corporation). Individual BAs and α-thujene were procured from PhytoLab GmbH & Co. (Vestenbergsgreuth, Germany) and Toronto Research Chemicals (Toronto, Canada), respectively. Fluoxymesterone was procured from LGC Standards (Wesel, Germany). HPLC-grade solvents were used for all the analysis (Merck Life Sciences Private Limited, Bengaluru, India). MilliQ plus (Millipore India Private Limited, Bengaluru, India) purified water was also employed.

Physicochemical Characterisation of BFQ-20

Colour and appearance of the extracts were noted manually. Particle size distribution of the powder and its tapped density were analysed by a Ro-Tap sieve analyser (Model-RX-29-10, WS Tyler, OH, U.S.A.) and bulk density apparatus (Model-951, Electronics India, Haryana, India), respectively.

Solubility and Particle Size Analysis

Solubility and particle size of the samples were evaluated as previously reported.²⁷⁾ Briefly, about 200 mg of sample was added to 100 mL deionized water and sonicated for 5 min using a bath sonicator (PCI Analytics, Mumbai, India), followed by centrifugation (3000 rpm for 2 min). The solution was then subjected to HPLC analysis and the soluble BA content was estimated. Particle size and zeta potential of the solution were then evaluated by a dynamic light scattering (DLS) method, employing Horiba SZ-100 particle size analyser (Horiba India Private Limited, Bengaluru, India).

In-Vitro Dissolution Study

In-vitro release study was performed, in triplicate, for BFQ-20 and U-BE under simulated gastric fluid (SGF) and simulated intestinal fluid (SIF) conditions, following the method of Ting et al.³⁰⁾ Briefly, 50 mg of sample was taken in a screw caped centrifuge tube and mixed with 5 mL SGF (3 g/L pepsin, 8.5 g/L NaCl; pH 3.0) and vortexed for 5 s followed by incubation at 37 °C for 0.5, 1 and 1.5 h. Intestinal condition was mimicked by further addition of 5 mL of SIF (10 g/L pancreatin in phosphate buffered saline (PBS), 3 g/L bile salts; pH 7.5) to the same test mixtures and further incubated for 2, 2.5, 3, 4, 5, and 6 h. Finally, the test mixture was centrifuged at 4500 rpm for 5 min and the supernatant was analysed for individual BA content and hence the percentage of bioaccessibility using below equation.

Accelerated Stability Study

Accelerated stability of BFQ-20 powder was conducted according to the guidelines of the International Conference on Harmonization (ICH) of technical requirements for the registration of pharmaceuticals for human use.³¹⁾ The samples (10 g packets) were incubated in a stability chamber (Thermolab Scientific Equipment’s Private Limited, Mumbai, India) kept at 40 ± 2 °C and 70 ± 5% relative humidity for a period of 6 months. Samples were retrieved at various time points (0, 1, 2, 3, and 6 months) and subjected to analysis for different physical and chemical parameters like colour, appearance, moisture, and the contents of BAs and α-thujene. The microbial parameters included total plate counts, yeast and mould, coliforms, Escherichia coli and Salmonella. Each analysis was carried out in triplicate.

Pharmacokinetics Study

Participants and Study Design

A total of 24 healthy volunteers of both sexes were screened; out of which 16 (aged between 20 to 58; 10 males and 6 females) eligible participants were randomized and completed the study as per the double-blinded, single-dose crossover design (Fig. 1). The number of participants were calculated using G power 3 software with a power of 80%, based on a previous study.³²⁾ Computer generated randomization was employed for randomisation.³³⁾ The study protocol was approved by a registered ethical committee and was performed as per the guidelines for Good Clinical Practices established by the Government of India (CTRI/2018/03/012753, dated 22/03/2018), in accordance with the Helsinki declaration. The participants were healthy, non-pregnant, non-smoking, non-alcoholic, and were not under any dietary supplements or medication. Individuals with active diseases were also excluded from the study. The procedure and objective of the study were detailed to the participants and received a written signed informed consent.

Fig. 1. Schematic Representation of the Generalized Protocol Adopted for the Randomized Double-Blinded, Placebo-Controlled, 2-Way Crossover Pharmacokinetic Study

Intervention

Selected participants were randomly assigned to one of the two treatment arms consisting of two single-dose treatments with either BFQ-20 or U-BE. A minimum of 14 d’ washout period was provided between the treatments. To investigate the relative bioavailability, each volunteer was given with a single dose of 400 mg of either BFQ-20 or U-BE, along with 200 ± 20 mL water to swallow. Boswellic acids and α-thujene contents of BFQ-20 and U-BE were given in Table 1. About 100 mg of microcrystalline cellulose (MCC) and maltodextrin in the ratio of 2 : 1 (w/w) was used as the filler for capsules, so that the total weight of each formulated and unformulated capsule remains at 550 ± 20 mg.

Table 1. Dose Details of U-BE and BFQ-20 Used for the Pharmacokinetics Study

Specification	KBA* (mg)	AKBA (mg)	αBA (mg)	βBA (mg)	AαBA (mg)	AβBA (mg)	Total boswellic acids (mg)	α-Thujene (mg)
U-BE	3.89	10.60	5.49	17.38	2.42	7.19	46.97	0.05
BFQ-20	1.00	10.50	1.50	4.60	0.61	1.90	20.11	3.04

* 11-Keto-β-boswellic acid (KBA), acetyl-11-keto-β-boswellic acid (AKBA), β-boswellic acid (βBA), acetyl-β-boswellic acid (AβBA), α-boswellic acid (αBA), and acetyl-α-boswellic acid (AαBA). The amount’s declared BAs are given in mg/100 mg.

Protocol and Sample Preparation for the Pharmacokinetic Study

Figure 1 shows the schematic representation of the study procedure. The volunteers were reported to the study centre by 7 to 8 a.m. under fasting. Using all aseptic precautions, an indwelling venous catheter was fixed in the forearm. About 2 mL each blood samples were collected in ethylenediaminetetra acetic acid (EDTA) tubes; before dosing (zero-time point) and at regular post-administration time intervals (1, 2, 4, 6, 8, and 24 h). The collected blood samples were centrifuged immediately (11950 × g for 10 min at 4 °C) to separate the plasma and stored at −20 °C for analysis. Acetonitrile was used to extract the BAs and α-thujene from plasma. Briefly, 1 mL of plasma was extracted with 1 mL ice-cold acetonitrile with vortex for 1 min and centrifuged (15 min, 9000 × g) at 4 °C, and the top layer was collected. The extraction was repeated three times and evaporated under a stream of nitrogen at 40 ± 2 °C. Using a mixture of acidified (0.1% (v/v) formic acid) acetonitrile/water (90 : 10, v/v) the residue was made up to 1 mL and filtered through a 0.22 µm syringe filter and analysed.

Ultra Performance Liquid Chromatography Coupled with Electrospray Ionisation Tandem Mass Spectrometry (UPLC-ESI-MS/MS)

For the detection, confirmation, and quantification of BAs in plasma, mass spectrometric analysis was performed in the negative single ion mode using Agilent Triple Quadrupole LC/MS 6460 series (Agilent Technologies, CA, U.S.A.). Separation of BAs was achieved with a Phenomenex column (100 × 3.0 mm; 2.6 µm) kept at 40 °C, and using the mobile phase system consisting of (A) 5 mm ammonium formate with 0.1% formic acid and (B) 0.1% formic acid in acetonitrile, at a flow rate of 0.5 mL/min. The molecular ions corresponding to the individual boswellic acids have been identified and confirmed by their UPLC profile and mass spectrometry, with the help of the corresponding analytical standards. The detected and confirmed ions were m/z 511.5 for AKBA, m/z 469.2 for KBA, m/z 455.3 for both αBA and βBA and m/z 497.2 for both AαBA and AβBA (Fig. 2). Fluoxymesterone was used as an internal standard. Data acquisition and integration of the peak areas were achieved using the software MassHunter version B 08.02. The range and linearity of the extraction efficiency were determined by spiking 10 ng/mL of BAs in plasma.

Fig. 2. (A) UPLC-MS Analysis to Quantify BAs in Plasma (a) Blank (b) Mixed Analytical Standard Solution (c) Plasma Sample Extract Collected after 3 h of Ingestion of BFQ-20; (B) Matrix-Matched Calibration Curves of BAs (KBA, AKBA, αBA, βBA, AαBA and AβBA) in Plasma

GC-MS/MS Analysis

GC-MS/MS method is good for the analysis of the volatile oil components like terpenes and terpenoids. Plasma concentration of α-thujene was measured by GC-MS/MS as follows. A gas chromatography system (Nexis GC-2030) coupled to a triple quadrupole mass spectrometer (Shimadzu TQ 8040 NX) was employed for the detection, confirmation, and quantification of α-thujene in plasma. A cyanopropyl dimethylpolysiloxane column (30 m × 0.32 mm; 1.8 µm) and helium carrier gas were used for the separation. The injector port temperature was kept at 250 °C and the column temperature was maintained at 65 °C for 5 min, then raised at a rate of 10 °C per minute to 210 °C. A tentative identification of the compound was performed on the basis of retention time and single ion mass spectra with the standard spectra available with the database library NIST (National Institute of Standards and Technology, U.S.A.). The main fragment ions detected for α-thujene (Molecular Formula: C₁₀H₁₆; Molecular weight: 136.23), in single ion mode were m/z: 93, 91, and 77, as per the method of Sereshti et al.³⁴⁾

In-Vivo Anti-inflammatory Activity

In-vivo anti-inflammatory activity of U-BE and BFQ-20 was evaluated using carrageenan-induced paw oedema method as described by Winter et al.³⁵⁾ Briefly, Sprague Dawley (SD) rats (150 to 200 g b. wt.) were divided into four groups (n = 5/group) and fasted overnight with ad libitum. On next day morning, the animals were randomised into group I (normal saline treated), Group II (treated with Diclofenac at 10 mg/kg b. wt. p.o.), Group III (treated with U-BE, 75 mg/kg b. wt. p.o.) and Group IV (treated with BFQ-20 at 75 mg/kg b. wt. p.o.). All the samples were prepared in saline solution for administration. Paw oedema was induced by subcutaneous injection of 1% carrageenan (100 µL) in normal saline solution into the left hind paw of rats, after 1 h post-administration. The paw volume was measured immediately before (basal) and after 5 h of injection using a plethysmometer.

Statistical Analysis

The data were analysed using SPSS software version 27 and presented as mean ± standard deviation. The significant difference in plasma pharmacokinetic parameters (area under the curve (AUC), C_max, T_max, T_1/2) was analysed using paired and independent t-test. The evaluation of anti-inflammatory effect was analysed using ANOVA followed by Dunnett’s test to estimate the differences between the groups. A ‘p’ value less than 0.05 was considered statistically significant. All data processing was performed using GraphPad Prism Version 8.0.

RESULTS

Boswellic acids standardised extract prepared by an ethanol-water extraction process of Boswellia serrata oleo-gum-resin (unformulated boswellia extract, U-BE), and its hybrid-hydrogel formulation (BFQ-20) were used for the pharmacokinetic study. The individual BAs and α-thujene contents of the extracts were given in Table 1. HPLC analysis revealed 46.97% of total BAs (sum of six boswellic acids, in which 10.60% accounted for AKBA) for U-BE. BFQ-20 on the other hand was found to contain only 20.11% of total BAs, with 10.50% AKBA. Gas chromatography analysis showed 0.05 and 3.04% α-thujene content in U-BE and BFQ-20, respectively. BFQ-20 was a water-dispersible creamish-brown powder with a tapped density of 0.67 g/mL; U-BE on the other hand was a light creamish water-insoluble powder. The solubility of BAs in BFQ-20 was found to be increased by about 58-times and representative image showing aqueous solubility is shown in Fig. 3A. The average particle size for initial BAs emulsion and BFQ-20 were 71.8 ± 4.6 and 183.6 ± 12.7 nm (Fig. 3B), respectively. Zeta potential of BAs emulsion and BFQ-20 were −5.6 and −53.2 ± 2.1 mV, respectively.

Fig. 3. (A) Digital Photograph of the Aqueous Solutions of BFQ-20 and U-BE; (B) Particle Size Distribution of BFQ-20

The inset figure shows the particle size distribution of boswellia extract emulsion.

In-Vitro Dissolution Study

In-vitro release study under SGF and SIF conditions showed a significant increase in the release of boswellic acids into solution, as demonstrated by the measurement of individual BAs content, from BFQ-20 compared to U-BE. This shows the enhancement in solubility of the boswellia extract upon formulation. Since the improvement in aqueous solubility of lipophilic components has been correlated with the bioaccessibility of these components, graphs were constructed to show the percentage of bioaccessibility of BAs vs. time, these graphs demonstrated a significant improvement in the bioaccessibility of BAs in BFQ-20, especially with a more than 6-fold increase in KBA and AKBA during 5 h of digestion (Fig. 4). A similar bioaccessibility were observed for other BAs also, with a slightly lower digestibility for AαBA and AβBA compared to KBA and AKBA (Fig. 4).

Fig. 4. In-Vitro Dissolution Profile of BFQ-20 and U-BE in Both Gastric (pH 3) and Intestinal (pH 7) pH Conditions

Accelerated Stability Study

The accelerated stability study at 40 ± 2 °C and at a relative humidity of 70 ± 5% for 6 months showed no significant changes (<5% deviation) for the physicochemical properties such as colour, appearance, bulk density, moisture content, and microbial load. The contents of BAs and α-thujene were also remain with 5% deviation, indicating its storage stability equivalent of 2 years under ambient conditions when kept in air-tight closed containers with less than 30 °C³¹⁾ (Table 2).

Table 2. Accelerated Stability of BFQ-20 as per ICH Guidelines at 40 ± 2 °C and RH 75 ± 5%

Parameters	Specification	0 month	1 month	2 months	3 months	6 months
Appearance	Free-flowing powder	Complies	Complies	Complies	Complies	Complies
Identification	HPTLC	Complies	Complies	Complies	Complies	Complies
Colour	Creamish brown	Complies	Complies	Complies	Complies	Complies
Tapped density	0.5–0.7 g/mL	0.67 g/mL	0.68 g/mL	0.66 g/mL	0.65 g/mL	0.67 g/mL
Boswellic acids content	18–20%	20.18%	20.16%	20.13%	20.02%	20.06%
AKBA	9.0–10.5%	10.52%	10.34%	10.31%	10.31%	10.16%
α-Thujene	2.5–3.5%	3.5%	3.4%	3.4%	3.2%	3.1%
Microbiology	US-FDA (BAM)
Total plate count^#	<1000 cfu/g	500 cfu/g	500 cfu/g	500 cfu/g	500 cfu/g	500 cfu/g
Yeast & mould^#	<100 cfu/g	<100 cfu/g	<100 cfu/g	<100 cfu/g	<100 cfu/g	<100 cfu/g
Salmonella^#	Absent/25 g	Absent/25 g	Absent/25 g	Absent/25 g	Absent/25 g	Absent/25 g
Escherichia coli^#	Absent/g	Absent/g	Absent/g	Absent/g	Absent/g	Absent/g
S. aureus^#	Absent/g	Absent/g	Absent/g	Absent/g	Absent/g	Absent/g

* NMT denotes ‘not more than’; # each value was presented as an average of three measurements.

Pharmacokinetics of BAs

UPLC-ESI-MS/MS is a reliable technique to detect, confirm and quantify individual BAs content in boswellia extracts and in biomatrices. The analytical methods used in the present study were based on the previous report by Gerbeth et al., and was validated as per ICH Q2(R1) guidelines.³⁶⁾ Acetonitrile-based sample preparation method provided an overall recovery of above >90% for individual boswellic acids, and about 88% for α-thujene. Linearity of the methods was determined by plotting the calibration curves with different concentrations of analytes (in the range 1 to 1000 ng/mL) versus the peak area. The calibration curves for individual analytes were linear with correlation coefficients >0.995 and limit of quantification (LOQ) of 1 ng/mL, indicating the validity and suitability of the methods employed for the quantification in plasma. The pharmacokinetic parameters, [maximum plasma concentration (C_max), post-administration time point at which maximum plasma concentration was observed (T_max), Time taken to reduce the maximum plasma concentration to half (T_1/2), and AUC], were deduced from the plasma concentration versus time plot for individual BAs and α-thujene.

The mean plasma concentration–time curve profile for U-BE and BFQ-20 are illustrated in Fig. 5, and the pharmacokinetic parameters are presented in Table 3. From the plasma concentration of various BAs (KBA, AKBA, αBA, βBA, AαBA, and AβBA), it was evident that BFQ-20 provided significantly higher concentration of BAs in plasma compared to U-BE, at all the post-administration time points (p < 0.0001). This was further clear from the pharmacokinetic properties C_max, T_max, AUC and T_1/2 for the individual boswellic acids (Fig. 5, Table 3). The C_max for various BAs from BFQ-20 demonstrated an increase of 2.94, 3.79, 2.59, 2.73, 3.23, and 1.59-fold respectively for KBA, AKBA, αBA, βBA, AαBA, and AβBA, compared to those for U-BE (p < 0.001). A similar comparison of AUC_0–24 h for individual BAs following the ingestion of U-BE and BFQ-20 revealed 4-fold increase for KBA, 6-fold for AKBA, 2.9-fold for αBA, 4.4-fold for βBA, 5.1-fold for AαBA, and 4.1-fold for AβBA, indicating a significant enhancement in relative bioavailability for each of the BAs from BFQ-20 (p < 0.001) (Fig. 5, Table 3). Moreover, the absorbed BAs from BFQ-20 was found to stay in the systemic circulation for longer duration, as evidenced from their T_1/2 values (Fig. 5, Table 3).

Fig. 5. Plasma Concentration–Time Plot for BAs (KBA, AKBA, αBA, βBA, AαBA, AβBA) Following the Ingestion of BFQ-20 and U-BE

Statistical analysis was completed using SPSS software version 27, and all the data points were presented as mean with standard deviation. Graphs were plotted using GraphPad Prism Version 8.0.

Table 3. Pharmacokinetic Parameters of the Boswellic Acids (BAs) and α-Thujene Following the Ingestion of U-BE and BFQ-20

BA’s*		C_max (ng/mL)	T_max (h)	T_1/2 (h)	AUC (ng ＊ h/mL)
KBA	BFQ-20	275.09 ± 117.76	6.53 ± 0.91	16.24 ± 1.12	3588.46 + 644.95
KBA	U-BE	93.50 ± 33.51	2.20 ± 0.56	8.52 ± 0.91	893.85 + 299.31
AKBA	BFQ-20	61.18 ± 26.79	4.27 ± 0.70	5.66 ± 0.87	298.92 + 71.06
AKBA	U-BE	16.12 ± 4.77	1.93 ± 0.25	3.86 ± 0.68	49.82 + 8.77
αBA	BFQ-20	94.90 ± 37.25	6.80 ± 1.01	16.46 ± 1.87	1154.05 + 231.09
αBA	U-BE	36.54 ± 14.12	4.27 ± 1.02	12.03 ± 1.24	404.29 + 210.88
βBA	BFQ-20	230.27 ± 59.28	5.07 ± 1.03	9.21 ± 0.99	2717.80 + 294.78
βBA	U-BE	84.22 ± 30.27	4.70 ± 0.83	7.18 ± 0.65	622.31 + 131.38
AαBA	BFQ-20	56.03 ± 31.77	6.53 ± 0.91	10.09 ± 1.84	558.22 + 134.34
AαBA	U-BE	17.34 ± 8.64	8.60 ± 0.82	6.3 ± 0.65	109.15 + 29.19
AβBA	BFQ-20	87.88 ± 28.64	7.07 ± 1.30	17.42 ± 1.98	1166.74 + 184.83
AβBA	U-BE	55.14 ± 20.46	4.27 ± 0.70	9.93 ± 1.12	286.45 + 55.00
Total BA	BFQ-20	729.22 ± 113.29	6.27 ± 0.74	15.53 ± 1.45	9484.17 + 767.82
Total BA	U-BE	254.21 ± 35.10	4.80 ± 1.04	8.86 ± 1.36	2365.87 + 346.89
α-Thujene	BFQ-20	68.80 ± 18.60	4.12 ± 0.38	16.24 ± 1.12	298.60 ± 35.48
α-Thujene	U-BE	3.65 ± 0.90	1.18 ± 0.12	8.50 ± 0.91	6.86 ± 2.31

* BA’s-boswellic acids; 11-keto-β-boswellic acid (KBA), acetyl-11-keto-β-boswellic acid (AKBA), β-boswellic acid (βBA), acetyl-β-boswellic acid (AβBA), α-boswellic acid (αBA), and acetyl-α-boswellic acid (AαBA). The data were presented as mean ± standard deviation.

Since all the six BAs have been shown to possess anti-inflammatory potential, we also constructed the plasma concentration verses time plot for the total boswellic acids in plasma (Fig. 6, Table 3). It was observed that BFQ-20 possesses 2.9-fold enhancement in absorption [C_max: 729.22 ± 113.29 vs. 254.21 ± 35.10 ng/mL], and 4-fold enhancement in the relative bioavailability of total BAs [AUC_0–24 h: (9484.17 ± 767.82 vs. 2365.87 ± 346.89 ng * h/mL], when compared with an equivalent dose of the unformulated standard extract (U-BE). Total BAs from BFQ-20 reached its maximum after 6.3 h post-administration, and the half-life was 15.5 h. For α-thujene, the C_max and AUC_0–24 h for the formulation BFQ-20 were found to be 68.80 ± 18.60 ng/mL and 298.60 ± 35.48 ng * h/mL, respectively, while those from the unformulated boswellia extract (U-BE) was negligibly small [C_max: 3.65 ± 0.90 ng/mL and AUC: 6.86 ± 2.31 ng * h/mL] (Fig. 7, Table 3).

Fig. 6. Plasma Concentration–Time Plot for the Total BAs Content in Plasma Following the Ingestion of U-BE and BFQ-20

Fig. 7. Plasma Concentration–Time Plot for the α-Thujene Content in Plasma Following the Ingestion of U-BE and BFQ-20

In-Vivo Anti-inflammatory Activity

The results showed about 42.0% inhibition of inflammation in Diclofenac and 38.0% inhibition in U-BE treated groups at 5th hour post-induction of oedema. One-way ANOVA analysis showed no statistically significant (p > 0.05) difference between Diclofenac and U-BE treatments (Fig. 8). However, BFQ-20 showed 70.5% inhibition which was significant (p < 0.05), compared to both Diclofenac and U-BE groups.

Fig. 8. (A) Photographs of Paw Oedema upon Treatment with BFQ-20 (75 mg/kg b. wt.) and U-BE (75 mg/kg b. wt.) in Comparison with Diclofenac at 5th Hour; (B) Percentage of Inhibition in Paw Oedema upon Treatment with Individual Components of AKBA Formulations at 75 mg/kg. b. wt. Dosage in Comparison with Diclofenac at 10 mg/kg b. wt. Orally

Adverse Events/Safety

The study did not show any significant adverse events or clinical signs due to U-BE and BFQ-20 supplementation.

DISCUSSION

The synergetic action of boswellic acids (BAs) and the essential oil composed of terpenes and terpenoids, mainly α-thujene (>60% (w/w)), was identified as the main principle responsible for the beneficial pharmacological effects of B. serrata oleo-gum resin.^2,37) But stable extracts containing both the BAs and essential oil, so-called full-spectrum extracts, have not been reported so far. Similarly, no information is available on the pharmacokinetics and bioavailability of the essential oil components of boswellia gum resin, though BAs have well been characterised to suffer from poor oral bioavailability due to hydrophobicity and low water solubility.^12,38) In the present contribution, we report the stability, solubility, and human pharmacokinetics of both BAs and α-thujene, following the ingestion of a natural self-emulsifying reversible hybrid-hydrogel (N’SERH) formulation of boswellia extract (BFQ-20) in comparison with the standardised unformulated boswellia extract (U-BE). Since fenugreek mucilage (galactomannan) and sunflower lecithin were employed, the formulation involved no synthetic excipients and can be considered as a ‘green’ food-grade formulation.

The hybrid-hydrogel form of boswellia extract (BFQ-20) was produced by impregnating lecithin based-emulsions of standardised B. serrata extract within the hydrogel scaffold followed by dehydration to powder state, with improved solubility of BAs (58-fold). The impregnation was clear from the particle size analysis which showed an increase from 71.8 ± 4.6 to 183.6 ± 12.7 nm when the emulsion is made into hybrid-hydrogel. There was also a corresponding enhancement in zeta potential, from −5.6 to −53.2 mV, indicating better stability of the hybrid hydrogel. Higher zeta potential corresponds to higher surface area and electrostatic repulsion which leads to lesser particle aggregation.³⁹⁾ The increase in the hydrodynamics diameter and zeta potential demonstrates the action of hydrogel scaffold as a ‘Trap’ for emulsions/liposomes/micelles during the storage and stomach conditions.

The in-vitro dissolution studies showed that the hybrid–hydrogel matrix improved the release pattern of solubilised boswellic acids in a sustained manner, and enhanced the bioaccessibility significantly, compared to U-BE (approx. 6-fold). Mucoadhesive nature of the fenugreek mucilage hydrogel helps longer contact time in the gastrointestinal tract which may lead to longer interaction of lipophilic components with enterocytes for better bioaccessibility. Generally, most of the lipophilic and lipid components may undergo hydrolysis during gastric digestion and form mixed micelles under the action of bile and bile salts. This process reduces the level of the accessible bioactives, (i.e., solubilized colloidal species in the lumen), for gastro-intestinal absorption.³⁰⁾ Relatively short elimination half-life has well established for standardised unformulated boswellia extracts.¹⁹⁾

Pharmacokinetics further demonstrated significantly enhanced absorption and relative bioavailability of all the six major boswellic acids and α-thujene from BFQ-20 compared to U-BE, as evident from the enhancement observed for their C_max and AUC. The total BAs absorption reached its maximum level after 6.3 h post-administration with a half-life of 15.5 h indicating the sustained intestinal release of emulsified and soluble particles of boswellia extracts from BFQ-20 for better absorption. There was 4- and 6-fold enhancement in the relative bioavailability of KBA and AKBA, respectively which were highly significant compared many previous reports. In a previous study, 1600 mg of a standardised B. serrata extract detected no major BAs except KBA in plasma, and produced no adverse effects.⁴⁰⁾ In another study, 3 g per day (786 mg ×4) for 10 d could provide a C_max of 51.20 and 160 ng/mL for AKBA and KBA, respectively.⁴¹⁾ In yet another study, 4 weeks of treatment at 2.40 g per day (approx. 800 mg ×3), could detect only AKBA at 20.50 ng/mL in plasma.²¹⁾ However, these doses, as high as 3 g per day, were well tolerated and produced no side effects. We also observed no side effects in this study.

Keto-boswellic acid (KBA) and acetyl keto-boswellic acid (AKBA) are identified as the most bioactive (anti-inflammatory and anticancer) boswellic acids. Molecular docking and in-vitro studies have identified 11-keto group to play critical role in the bioactivities of boswellic acids. In a molecular docking study of various BAs with the LOX enzyme, AKBA revealed most favourable interactions with the best dock score and binding free energy. The observed order of binding was AKBA ≫ KBA ≫ βBA ≫ αBA.⁴²⁾ In an in-vitro study, replacement of keto group by a methylene group or reduction into an alcohol group has been reported to reduce the activity significantly.⁴³⁾ Other in-vitro studies to compare the anticancer activities of individual boswellic acids have also shown better activity for β-boswellic acids,⁴⁴⁾ with AKBA being the most anti-proliferative, anti-metastatic and apoptotic.^45,46) In BFQ-20, the relative concentrations of these BAs were observed as AKBA-10.50%, KBA-1.00%, αBA-1.50%, βBA-4.60%, AαBA-0.61%, AβBA-1.90%, indicating the possibility for better anti-inflammatory effect.

The pharmacokinetic parameters deduced in the present study reveals the significance of the present formulation among the various formulations reported so far for human supplementation. Meins et al. reported a nanoemulsion of boswellia extract using polysorbate to increase the bioavailability of boswellic acids in rodents.²⁴⁾ Riva et al. reported that the phosphatidyl complex of boswellia extract can increase the bioavailability.³²⁾ However, these formulations were devoid of the most active volatile part of the oleo-gum resin of boswellia, since such formulations cannot stabilize the volatile molecules. It was already shown that the volatile fractions can increase the bioavailability of the non-volatile molecules and can have synergetic anti-inflammatory effect, as in the case of curcumin and turmerones.⁴⁷⁾ Here comes the significance of BFQ-20 as a hybrid-hydrogel where lecithin-based emulsions of boswellia extract was impregnated in the hydrogel matrix and stabilised. It is also a green, full-spectrum formulation of boswellia extract composed of essential oil and lecithin-rich sunflower oil, based on the previous reports that the ingestion of boswellia extract along with fat food can support the bioavailability of boswellic acids.²⁰⁾

Further investigation on the anti-inflammatory potential of BFQ-20 employing the well-validated carrageenan-induced paw oedema rat model revealed a positive impact of enhancement in bioavailability on the in-vivo anti-inflammatory effect, as observed from the percentage of inhibition of paw oedema (38.0 vs. 70.5%). Carrageenan is a strong chemical that can cause the release of inflammatory and pro-inflammatory mediators (prostaglandins, leukotrienes, histamine, bradykinin, TNF-α, etc.).⁴⁸⁾ Several preclinical studies have already established the positive effect of B. Serratta extracts in carrageenan induced inflammatory model of rats,^49–51) and hence this model serve as a simple and reliable method for the correlation of bioavailability and efficacy.

In 2012, Krishnakumar et al. reported the use of fenugreek mucilage, (galactomannan soluble dietary fiber), for the first time, as a mucoadhesive hydrogel matrix for the oral delivery of hydrophobic curcumin with improved solubility and bioavailability (FenuMat^®).⁵²⁾ Later, fenugreek mucilage has been found to be an ideal platform to trap the relatively unstable nano structures like liposomes, micelle and emulsions within the pockets created by the three dimensional network of galactomannan chains to form hybrid-hydrogels.^26,27) Upon dehydration, these hydrogels can be converted to powder form and upon rehydration or in the gastrointestinal tract, the powder can be swelled back to hydrogel form and further into a viscous solution state, indicating its reversible nature. Thus, fenugreek galactomannan-based hybrid-hydrogel systems emerged as means to address the inherent problems of nanoemulsions, liposomes and micelles, such as their tendency to aggregate, especially under gastrointestinal conditions, leading to the seepage of bioactive molecules.⁵³⁾ Our previous studies on resveratrol, fisetin, and quercetin have also demonstrated the improved solubility, stability, and bioavailability of the corresponding hybrid-hydrogel systems.^25–28)

CONCLUSION

In conclusion, the present study demonstrated a stable, soluble and bioavailable formulation of a full-spectrum extract of B. serrata oleo-gum resin [containing both non-volatile (BAs) and volatile (α-thujene) bioactives], employing a natural self-emulsifying reversible hybrid-hydrogel (N’SERH) technology (FenuMat^®) for the first time. The formulation (BFQ-20) was developed from a hydro-ethanolic extract of B. serrata oleo-gum-resin, and standardised to a total boswellic acid content (sum of six boswellic acids KBA, AKBA, αBA, βBA, AαBA, and AβBA) as not less than 18% (w/w). The formulation contained 3.04% α-thujene and 10.50% (w/w) of AKBA, (the most bioactive terpene and boswellic acid in B. serrata oleo-gum-resin). The hybrid-hydrogel formulation showed enhanced solubility (58-fold), stability and sustained release of boswellia emulsions under gastrointestinal conditions, which effectively increased the relative bioavailability and elimination half-life of boswellic acids and α-thujene. Yet another important aspect of this study is the use of fenugreek mucilage as the hydrogel scaffold, which is a non-genetically modified, allergen-free functional prebiotic fiber. Considering the clean label status of this green formulation and interesting pharmacokinetic parameters, we recommend future clinical research for potential human benefits.

Acknowledgments

The authors thank Leads Clinical Research & Bioservices Private Limited, Bangalore, India, for the coordination of the human pharmacokinetic study. We acknowledge Dr. Umesh at Akay Bioactives for his support in the preparation of manuscript.

Funding

This research is financially supported by Akay Natural Ingredients Private Limited, Kochi, Kerala, India.

Author Contributions

Conceptualisation, Investigation, Supervision, Writing, Review, and Editing: K.I.M.; Data curation, Methodology, Former analysis: A.J., M.B.A. & J.N.M.; Project administration, M.B.A.

Conflict of Interest

The authors declare no conflict of interest.

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